SummaryPantoea agglomerans pv. gypsophilae (Pag) elicits galls on gypsophila and a hypersensitive response on beet, whereas P. agglomerans pv. betae (Pab) induces galls on both beet and gypsophila. The pathogenicity of both pathovars is dependent on the presence of a plasmid harbouring type III secretion system (TTSS) components and effectors. The HsvG TTSS effectors of Pag (HsvG-Pag) and Pab (HsvGPab) determine the host specificity of both pathovars on gypsophila. Here we describe a novel HsvG homologue, HsvB, which determines the host specificity of Pag and Pab on beet. HsvG requires two direct amino acid repeats for pathogenicity on gypsophila, whereas one repeat in HsvB is sufficient for pathogenicity on beet. Exchanging repeats between HsvG-Pag and HsvB-Pab resulted in a switch of host specificities. Transient expression of GFP-HsvG or GFP-HsvB fusions in gypsophila, beet or melon leaves showed that HsvG and HsvB were localized to the nuclei of host and non-host plants. A yeast one-hybrid assay revealed that a single repeat of HsvG or HsvB was sufficient to activate transcription. By employing random binding-site selection and gel-shift assay HsvG was demonstrated to be a double-stranded DNA-binding protein with an ACACC/aAA consensus binding site. These results suggest that HsvG and HsvB are host-specificity determinants and bear the potential to affect the host transcriptional machinery.

Reliable detection and identification of plant pathogens are essential for disease control strategies. Diagnostic methods commonly used to detect plant pathogens have limitations such as requirement of prior knowledge of the genome sequence, low sensitivity and a restricted ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled determination of total nucleic acid content in biological samples. The possibility of using the single‐molecule sequencing platform of Oxford Nanopore as a general method for diagnosis of plant diseases was examined. It was tested by sequencing DNA or RNA isolated from tissues with symptoms from plants of several families inoculated with known pathogens (e.g. bacteria, viruses, fungi, phytoplasma). Additionally, samples of groups of 200 seeds containing one infected seed of each of two or three pathogens, as well as samples with symptoms but unidentified pathogens were tested. Sequencing results were analysed with Nanopore data analysis tools. In all the inoculated plants, pathogens were identified in real time within 1–2 h of running the Nanopore sequencer and were classified to the species or genus level. DNA sequencing or direct RNA sequencing of samples with unidentified disease agents were validated by conventional diagnostic procedures (e.g. PCR, ELISA, Koch test), which supported the results obtained by Nanopore sequencing. The advantages of this technology include: long read lengths, fast run times, portability, low cost and the possibility of use in every laboratory. This study indicates that adoption of the Nanopore platform will be greatly advantageous for routine laboratory diagnosis.

Pantoea agglomerans, a widespread epiphytic bacterium, has evolved into a hypersensitive response and pathogenicity (hrp)-dependent and host-specific gall-forming pathogen by the acquisition of a pathogenicity plasmid containing a type III secretion system (T3SS) and its effectors (T3Es). Pantoea agglomerans pv. betae (Pab) elicits galls on beet (Beta vulgaris) and gypsophila (Gypsophila paniculata), whereas P. agglomerans pv. gypsophilae (Pag) incites galls on gypsophila and a hypersensitive response (HR) on beet. Draft genome sequences were generated and employed in combination with a machine-learning approach and a translocation assay into beet roots to identify the pools of T3Es in the two pathovars. The genomes of the sequenced Pab4188 and Pag824-1 strains have a similar size (∼5 MB) and GC content (∼55%). Mutational analysis revealed that, in Pab4188, eight T3Es (HsvB, HsvG, PseB, DspA/E, HopAY1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on beet and gypsophila. In Pag824-1, nine T3Es (HsvG, HsvB, PthG, DspA/E, HopAY1, HopD1, HopX2, HopAF1 and HrpK) contribute to pathogenicity on gypsophila, whereas the PthG effector triggers HR on beet. HsvB, HsvG, PthG and PseB appear to endow pathovar specificities to Pab and Pag, and no homologous T3Es were identified for these proteins in other phytopathogenic bacteria. Conversely, the remaining T3Es contribute to the virulence of both pathovars, and homologous T3Es were found in other phytopathogenic bacteria. Remarkably, HsvG and HsvB, which act as host-specific transcription factors, displayed the largest contribution to disease development.

HrpL, an alternative sigma factor, activates the transcription of the Hrp regulon by its binding to a common "hrp box" promoter. Based on computational techniques, the hrp box previously was defined as a consensus bipartite cis element, 5'-GGAACC-N(15-16)-CCACNNA-3'. The present report combines a quantitative in vivo assay for measuring Hrp promoter activity with site-specific mutagenesis to analyze the effect of consensus and nonconsensus nucleotides on promoter activity. The analysis was carried out with Hop effectors of the tumorigenic bacterium Pantoea agglomerans pv. gypsophilae, in which HrpL is indispensable for gall formation. Mutational analysis indicates that the hrp box consensus can be divided into crucial and noncrucial nucleotides. The first 5 nucleotides (nt) of the--35 consensus motif (GGAAC) and the 3 nt of the--10 motif (ACNNA) are crucial, whereas other consensus and adjacent nonconsensus nucleotides exert a significant effect on the promoter's strength. With spacing of 13 or 17 nt between the two motifs, significant activity was still retained. Gel shift assays indicated that deletion of GG from the--35 consensus motif eliminated HrpL binding, whereas mutations in the--10 consensus motif or modification of the spacing, which eliminates promoter activity, did not elicit any effect. The degeneracy in Hrp promoters of four hrp and type III effector genes of P agglomerans pv. gypsophilae indicated significant differences in promoter activity, whereas increasing the promoter strength of the Hop effector, HsvG, resulted in overexpression of gall formation.