Genetic Loci of Plant Pathogenic Dickeya solani IPO 2222 Expressed in Contact with Weed-Host Bittersweet Nightshade (Solanum dulcamara L.) Plants.

Dickeya solani, belonging to the Soft Rot Pectobacteriaceae, are aggressive necrotrophs, exhibiting both a wide geographic distribution and a wide host range that includes many angiosperm orders, both dicot and monocot plants, cultivated under all climatic conditions. Little is known about the infection strategies D. solani employs to infect hosts other than potato (Solanum tuberosum L.). Our earlier study identified D. solani Tn5 mutants induced exclusively by the presence of the weed host S. dulcamara. The current study assessed the identity and virulence contribution of the selected genes mutated by the Tn5 insertions and induced by the presence of S. dulcamara. These genes encode proteins with functions linked to polyketide antibiotics and polysaccharide synthesis, membrane transport, stress response, and sugar and amino acid metabolism. Eight of these genes, encoding UvrY (GacA), tRNA guanosine transglycosylase Tgt, LPS-related WbeA, capsular biosynthesis protein VpsM, DltB alanine export protein, glycosyltransferase, putative transcription regulator YheO/PAS domain-containing protein, and a hypothetical protein, were required for virulence on S. dulcamara plants. The implications of D. solani interaction with a weed host, S. dulcamara, are discussed.

A natural single nucleotide mutation in the small regulatory RNA ArcZ of Dickeya solani switches off the antimicrobial activities against yeast and bacteria.

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.

The integration host factor regulates multiple virulence pathways in bacterial pathogen Dickeya zeae MS2.

Dickeya zeae is an aggressive bacterial phytopathogen that infects a wide range of host plants. It has been reported that integration host factor (IHF), a nucleoid-associated protein consisting of IHFα and IHFß subunits, regulates gene expression by influencing nucleoid structure and DNA bending. To define the role of IHF in the pathogenesis of D. zeae MS2, we deleted either and both of the IHF subunit encoding genes ihfA and ihfB, which significantly reduced the production of cell wall-degrading enzymes (CWDEs), an unknown novel phytotoxin and the virulence factor-modulating (VFM) quorum-sensing (QS) signal, cell motility, biofilm formation, and thereafter the infection ability towards both potato slices and banana seedlings. To characterize the regulatory pathways of IHF protein associated with virulence, IHF binding sites (consensus sequence 5'-WATCAANNNNTTR-3') were predicted and 272 binding sites were found throughout the genome. The expression of 110 tested genes was affected by IHF. Electrophoretic mobility shift assay (EMSA) showed direct interaction of IhfA protein with the promoters of vfmE, speA, pipR, fis, slyA, prtD, hrpL, hecB, hcp, indA, hdaA, flhD, pilT, gcpJ, arcA, arcB, and lysR. This study clarified the contribution of IHF in the pathogenic process of D. zeae by controlling the production of VFM and putrescine QS signals, phytotoxin, and indigoidine, the luxR-solo system, Fis, SlyA, and FlhD transcriptional regulators, and secretion systems from type I to type VI. Characterization of the regulatory networks of IHF in D. zeae provides a target for prevention and control of plant soft rot disease.

Genomic and Functional Dissections of Dickeya zeae Shed Light on the Role of Type III Secretion System and Cell Wall-Degrading Enzymes to Host Range and Virulence.

Dickeya zeae is a worldwide destructive pathogen that causes soft rot diseases on various hosts such as rice, maize, banana, and potato. The strain JZL7 we recently isolated from clivia represents the first monocot-specific D. zeae and also has reduced pathogenicity compared to that of other D. zeae strains (e.g., EC1 and MS2). To elucidate the molecular mechanisms underlying its more restricted host range and weakened pathogenicity, we sequenced the complete genome of JZL7 and performed comparative genomic and functional analyses of JZL7 and other D. zeae strains. We found that, while having the largest genome among D. zeae strains, JZL7 lost almost the entire type III secretion system (T3SS), which is a key component of the virulence suite of many bacterial pathogens. Importantly, the deletion of T3SS in MS2 substantially diminished the expression of most type III secreted effectors (T3SEs) and MS2's pathogenicity on both dicots and monocots. Moreover, although JZL7 and MS2 share almost the same repertoire of cell wall-degrading enzymes (CWDEs), we found broad reduction in the production of CWDEs and expression levels of CWDE genes in JZL7. The lower expression of CWDEs, pectin lyases in particular, would probably make it difficult for JZL7 to break down the cell wall of dicots, which is rich in pectin. Together, our results suggest that the loss of T3SS and reduced CWDE activity together might have contributed to the host specificity and virulence of JZL7. Our findings also shed light on the pathogenic mechanism of Dickeya and other soft rot Pectobacteriaceae species in general. IMPORTANCE Dickeya zeae is an important, aggressive bacterial phytopathogen that can cause severe diseases in many crops and ornamental plants, thus leading to substantial economic losses. Strains from different sources showed significant diversity in their natural hosts, suggesting complicated evolution history and pathogenic mechanisms. However, molecular mechanisms that cause the differences in the host range of D. zeae strains remain poorly understood. This study carried out genomic and functional dissections of JZL7, a D. zeae strain with restricted host range, and revealed type III secretion system (T3SS) and cell wall-degrading enzymes (CWDEs) as two major factors contributing to the host range and virulence of D. zeae, which will provide a valuable reference for the exploration of pathogenic mechanisms in other bacteria and present new insights for the control of bacterial soft rot diseases on crops.

Influence of glucose on swarming and quorum sensing of Dickeya solani.

Dickeya solani is a pathogen most frequently responsible for infecting potato plants in Europe. As in the case of most plant pathogens, its ability to colonize and invade the host depends on chemotaxis and motility. The coordinated movement of Dickeya over solid surfaces is governed by a quorum sensing mechanism. In D. solani motility is regulated by ExpI-ExpR proteins, homologous to luxI-luxR system from Vibrio fisheri, in which N-acyl-homoserine lactones (AHLs) serve as signaling molecules. Moreover, in many Gram-negative bacteria motility is coupled with central metabolism via carbon catabolite repression. This enables them to reach more nutrient-efficient niches. The aim of this study was to analyze the swarming motility of D. solani depending on the volume of the medium in the cultivation plate and glucose content. We show that the ability of this bacterium to move is strictly dependent on both these factors. Moreover, we analyze the production of AHLs and show that the quorum sensing mechanism in D. solani is also influenced by the availability of glucose in the medium and that the distribution of these signaling molecules are different depending on the volume of the medium in the plate.

Design and comparative characterization of RecA variants.

RecA plays a central role in DNA repair and is a main actor involved in recombination and activation of the SOS response. It is also used in the context of biotechnological applications in recombinase polymerase isothermal amplification (RPA). In this work, we studied the biological properties of seven RecA variants, in particular their recombinogenic activity and their ability to induce the SOS response, to better understand the structure-function relationship of RecA and the effect of combined mutations. We also investigated the biochemical properties of RecA variants that may be useful for the development of biotechnological applications. We showed that Dickeya dadantii RecA (DdRecA) had an optimum strand exchange activity at 30 °C and in the presence of a dNTP mixture that inhibited Escherichia coli RecA (EcRecA). The differences between the CTD and C-tail of the EcRecA and DdRecA domains could explain the altered behaviour of DdRecA. D. radiodurans RecA (DrRecA) was unable to perform recombination and activation of the SOS response in an E. coli context, probably due to its inability to interact with E. coli recombination accessory proteins and SOS LexA repressor. DrRecA strand exchange activity was totally inhibited in the presence of chloride ions but worked well in acetate buffer. The overproduction of Pseudomonas aeruginosa RecA (PaRecA) in an E. coli context was responsible for a higher SOS response and defects in cellular growth. PaRecA was less inhibited by the dNTP mixture than EcRecA. Finally, the study of three variants, namely, EcPa, EcRecAV1 and EcRecAV2, that contained a combination of mutations that, taken independently, are described as improving recombination, led us to raise new hypotheses on the structure-function relationship and on the monomer-monomer interactions that perturb the activity of the protein as a whole.

An uncommon [K+(Mg2+)2] metal ion triad imparts stability and selectivity to the Guanidine-I riboswitch.

The widespread ykkC-I riboswitch class exemplifies divergent riboswitch evolution. To analyze how natural selection has diversified its versatile RNA fold, we determined the X-ray crystal structure of the Burkholderia sp. TJI49 ykkC-I subtype-1 (Guanidine-I) riboswitch aptamer domain. Differing from the previously reported structures of orthologs from Dickeya dadantii and Sulfobacillus acidophilus, our Burkholderia structure reveals a chelated K+ ion adjacent to two Mg2+ ions in the guanidine-binding pocket. Thermal melting analysis shows that K+ chelation, which induces localized conformational changes in the binding pocket, improves guanidinium-RNA interactions. Analysis of ribosome structures suggests that the [K+(Mg2+)2] ion triad is uncommon. It is, however, reminiscent of metal ion clusters found in the active sites of ribozymes and DNA polymerases. Previous structural characterization of ykkC-I subtype-2 RNAs, which bind the effector ligands ppGpp and PRPP, indicate that in those paralogs, an adenine responsible for K+ chelation in the Burkholderia Guanidine-I riboswitch is replaced by a pyrimidine. This mutation results in a water molecule and Mg2+ ion binding in place of the K+ ion. Thus, our structural analysis demonstrates how ion and solvent chelation tune divergent ligand specificity and affinity among ykkC-I riboswitches.

Identification of Three Type II Toxin-Antitoxin Systems in Model Bacterial Plant Pathogen Dickeya dadantii 3937.

Type II toxin-antitoxin (TA) systems are genetic elements usually encoding two proteins: a stable toxin and an antitoxin, which binds the toxin and neutralizes its toxic effect. The disturbance in the intracellular toxin and antitoxin ratio typically leads to inhibition of bacterial growth or bacterial cell death. Despite the fact that TA modules are widespread in bacteria and archaea, the biological role of these systems is ambiguous. Nevertheless, a number of studies suggests that the TA modules are engaged in such important processes as biofilm formation, stress response or virulence and maintenance of mobile genetic elements. The Dickeya dadantii 3937 strain serves as a model for pathogens causing the soft-rot disease in a wide range of angiosperm plants. Until now, several chromosome-encoded type II TA systems were identified in silico in the genome of this economically important bacterium, however so far only one of them was experimentally validated. In this study, we investigated three putative type II TA systems in D. dadantii 3937: ccdAB2Dda, phd-docDda and dhiTA, which represents a novel toxin/antitoxin superfamily. We provide an experimental proof for their functionality in vivo both in D. dadantii and Escherichia coli. Finally, we examined the prevalence of those systems across the Pectobacteriaceae family by a phylogenetic analysis.

PacBio-Based Protocol for Bacterial Genome Assembly.

Acquisition of high-quality bacterial genomes is fundamental, while having in mind investigation of subtitle intraspecies variation in addition to development of sensitive species-specific tools for detection and identification of the pathogens. In this view, Pacific Biosciences technology seems highly tempting taking into consideration over 10,000 bp length of the generated reads. In this work, we describe a bacterial genome assembly pipeline based on open-source software that might be handled also by non-bioinformaticians interested in transformation of sequencing data into reliable biological information. With the use of this method, we successfully closed six Dickeya solani genomes, while the assembly process was run just on a slightly improved desktop computer.

Comparative Genomics, from the Annotated Genome to Valuable Biological Information: A Case Study.

High availability of fast, cheap, and high-throughput next generation sequencing techniques resulted in acquisition of numerous de novo sequenced and assembled bacterial genomes. It rapidly became clear that digging out useful biological information from such a huge amount of data presents a considerable challenge. In this chapter we share our experience with utilization of several handy open source comparative genomic tools. All of them were applied in the studies focused on revealing inter- and intraspecies variation in pectinolytic plant pathogenic bacteria classified to Dickeya solani and Pectobacterium parmentieri. As the described software performed well on the species within the Pectobacteriaceae family, it presumably may be readily utilized on some closely related taxa from the Enterobacteriaceae family. First of all, implementation of various annotation software is discussed and compared. Then, tools computing whole genome comparisons including generation of circular juxtapositions of multiple sequences, revealing the order of synteny blocks or calculation of ANI or Tetra values are presented. Besides, web servers intended either for functional annotation of the genes of interest or for detection of genomic islands, plasmids, prophages, CRISPR/Cas are described. Last but not least, utilization of the software designed for pangenome studies and the further downstream analyses is explained. The presented work not only summarizes broad possibilities assured by the comparative genomic approach but also provides a user-friendly guide that might be easily followed by nonbioinformaticians interested in undertaking similar studies.

Genomic diversity and organization of complex polysaccharide biosynthesis clusters in the genus Dickeya.

The pectinolytic genus Dickeya (formerly Erwinia chrysanthemi) comprises numerous pathogenic species which cause diseases in various crops and ornamental plants across the globe. Their pathogenicity is governed by complex multi-factorial processes of adaptive virulence gene regulation. Extracellular polysaccharides and lipopolysaccharides present on bacterial envelope surface play a significant role in the virulence of phytopathogenic bacteria. However, very little is known about the genomic location, diversity, and organization of the polysaccharide and lipopolysaccharide biosynthetic gene clusters in Dickeya. In the present study, we report the diversity and structural organization of the group 4 capsule (G4C)/O-antigen capsule, putative O-antigen lipopolysaccharide, enterobacterial common antigen, and core lipopolysaccharide biosynthesis clusters from 54 Dickeya strains. The presence of these clusters suggests that Dickeya has both capsule and lipopolysaccharide carrying O-antigen to their external surface. These gene clusters are key regulatory components in the composition and structure of the outer surface of Dickeya. The O-antigen capsule/group 4 capsule (G4C) coding region shows a variation in gene content and organization. Based on nucleotide sequence homology in these Dickeya strains, two distinct groups, G4C group I and G4C group II, exist. However, comparatively less variation is observed in the putative O-antigen lipopolysaccharide cluster in Dickeya spp. except for in Dickeya zeae. Also, enterobacterial common antigen and core lipopolysaccharide biosynthesis clusters are present mostly as conserved genomic regions. The variation in the O-antigen capsule and putative O-antigen lipopolysaccharide coding region in relation to their phylogeny suggests a role of multiple horizontal gene transfer (HGT) events. These multiple HGT processes might have been manifested into the current heterogeneity of O-antigen capsules and O-antigen lipopolysaccharides in Dickeya strains during its evolution.

Structure-function analysis of pectate lyase Pel3 reveals essential facets of protein recognition by the bacterial type 2 secretion system.

The type II secretion system (T2SS) transports fully folded proteins of various functions and structures through the outer membrane of Gram-negative bacteria. The molecular mechanisms of substrate recruitment by T2SS remain elusive but a prevailing view is that the secretion determinants could be of a structural nature. The phytopathogenic γ-proteobacteria, Pectobacterium carotovorum and Dickeya dadantii, secrete similar sets of homologous plant cell wall degrading enzymes, mainly pectinases, by similar T2SSs, called Out. However, the orthologous pectate lyases Pel3 and PelI from these bacteria, which share 67% of sequence identity, are not secreted by the counterpart T2SS of each bacterium, indicating a fine-tuned control of protein recruitment. To identify the related secretion determinants, we first performed a structural characterization and comparison of Pel3 with PelI using X-ray crystallography. Then, to assess the biological relevance of the observed structural variations, we conducted a loop-substitution analysis of Pel3 combined with secretion assays. We showed that there is not one element with a definite secondary structure but several distant and structurally flexible loop regions that are essential for the secretion of Pel3 and that these loop regions act together as a composite secretion signal. Interestingly, depending on the crystal contacts, one of these key secretion determinants undergoes disorder-to-order transitions that could reflect its transient structuration upon the contact with the appropriate T2SS components. We hypothesize that such T2SS-induced structuration of some intrinsically disordered zones of secretion substrates could be part of the recruitment mechanism used by T2SS.

The nucleoid-associated protein IHF acts as a 'transcriptional domainin' protein coordinating the bacterial virulence traits with global transcription.

Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαß heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a 'transcriptional domainin' protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.

Genomic divergence between Dickeya zeae strain EC2 isolated from rice and previously identified strains, suggests a different rice foot rot strain.

Rice foot rot caused by Dickeya zeae is an important bacterial disease of rice worldwide. In this study, we identified a new strain EC2 from rice in Guangdong province, China. This strain differed from the previously identified strain from rice in its biochemical characteristics, pathogenicity, and genomic constituents. To explore genomic discrepancies between EC2 and previously identified strains from rice, a complete genome sequence of EC2 was obtained and used for comparative genomic analyses. The complete genome sequence of EC2 is 4,575,125 bp in length. EC2 was phylogenetically closest to previously identified Dickeya strains from rice, but not within their subgroup. In terms of secretion systems, genomic comparisons revealed that EC2 harbored only type I (T1SS), typeⅡ (T2SS), and type VI (T6SS) secretion systems. The flagella cluster of this strain possessed specific genomic characteristics like other D. zeae strains from Guangdong and from rice; within this locus, the genetic diversity among strains from rice was much lower than that of within strains from non-rice hosts. Unlike other strains from rice, EC2 lost the zeamine cluster, but retained the clustered regularly interspaced short palindromic repeats-1 (CRISPR-1) array. Compared to the other D. zeae strains containing both exopolysaccharide (EPS) and capsular polysaccharide (CPS) clusters, EC2 harbored only the CPS cluster, while the other strains from rice carried only the EPS cluster. Furthermore, we found strain MS1 from banana, carrying both EPS and CPS clusters, produced significantly more EPS than the strains from rice, and exhibited different biofilm-associated phenotypes. Comparative genomics analyses suggest EC2 likely evolved through a pathway different from the other D. zeae strains from rice, producing a new type of rice foot rot pathogen. These findings emphasize the emergence of a new type of D. zeae strain causing rice foot rot, an essential step in the early prevention of this rice bacterial disease.

Antibacterial activity and mode of action of potassium tetraborate tetrahydrate against soft-rot bacterial plant pathogens.

Bacterial soft rot caused by the bacteria Dickeya and Pectobacterium is a destructive disease of vegetables, as well as ornamental plants. Several management options exist to help control these pathogens. Because of the limited success of these approaches, there is a need for the development of alternative methods to reduce losses. In this study, we evaluated the effect of potassium tetraborate tetrahydrate (PTB) on the growth of six Dickeya and Pectobacterium spp. Disc diffusion assays showed that Dickeya spp. and Pectobacterium spp. differ in their sensitivity to PTB. Spontaneous PTB-resistant mutants of Pectobacterium were identified and further investigation of the mechanism of PTB resistance was conducted by full genome sequencing. Point mutations in genes cpdB and supK were found in a single Pectobacterium atrosepticum PTB-resistant mutant. Additionally, point mutations in genes prfB (synonym supK) and prmC were found in two independent Pectobacterium brasiliense PTB-resistant mutants. prfB and prmC encode peptide chain release factor 2 and its methyltransferase, respectively. We propose the disruption of translation activity due to PTB leads to Pectobacterium growth inhibition. The P. atrosepticum PTB-resistant mutant showed altered swimming motility. Disease severity was reduced for P. atrosepticum-inoculated potato stems sprayed with PTB. We discuss the potential risk of selecting for bacterial resistance to this chemical.

Comparative genomics and pangenome-oriented studies reveal high homogeneity of the agronomically relevant enterobacterial plant pathogen Dickeya solani.

BACKGROUND: Dickeya solani is an important plant pathogenic bacterium causing severe losses in European potato production. This species draws a lot of attention due to its remarkable virulence, great devastating potential and easier spread in contrast to other Dickeya spp. In view of a high need for extensive studies on economically important soft rot Pectobacteriaceae, we performed a comparative genomics analysis on D. solani strains to search for genetic foundations that would explain the differences in the observed virulence levels within the D. solani population. RESULTS: High quality assemblies of 8 de novo sequenced D. solani genomes have been obtained. Whole-sequence comparison, ANIb, ANIm, Tetra and pangenome-oriented analyses performed on these genomes and the sequences of 14 additional strains revealed an exceptionally high level of homogeneity among the studied genetic material of D. solani strains. With the use of 22 genomes, the pangenome of D. solani, comprising 84.7% core, 7.2% accessory and 8.1% unique genes, has been almost completely determined, suggesting the presence of a nearly closed pangenome structure. Attribution of the genes included in the D. solani pangenome fractions to functional COG categories showed that higher percentages of accessory and unique pangenome parts in contrast to the core section are encountered in phage/mobile elements- and transcription- associated groups with the genome of RNS 05.1.2A strain having the most significant impact. Also, the first D. solani large-scale genome-wide phylogeny computed on concatenated core gene alignments is herein reported. CONCLUSIONS: The almost closed status of D. solani pangenome achieved in this work points to the fact that the unique gene pool of this species should no longer expand. Such a feature is characteristic of taxa whose representatives either occupy isolated ecological niches or lack efficient mechanisms for gene exchange and recombination, which seems rational concerning a strictly pathogenic species with clonal population structure. Finally, no obvious correlations between the geographical origin of D. solani strains and their phylogeny were found, which might reflect the specificity of the international seed potato market.

Development of lateral flow assay combined with recombinase polymerase amplification for highly sensitive detection of Dickeya solani.

Dickeya solani, one of the most significant bacterial pathogens, infects potato plants, resulting in severe economic damage. In this study, a lateral flow assay (LFA) combined with isothermal DNA amplification was developed for rapid, specific, and sensitive diagnosis of the potato blackleg disease caused by D. solani. Recombinase polymerase amplification (RPA) was chosen for this purpose. Five primer pairs specific to different regions of the D. solani genome were designed and screened. A primer pair providing correct recognition of the target sequence was aligned with the SOL-C region specific to D. solani and flanked by fluorescein (forward primer) and biotin (reverse primer). Lateral flow test strips were constructed to detect DNA amplicons. The RPA-LFA demonstrated a detection limit equal to 14,000 D. solani colony-forming units per gram of potato tuber. This assay provided sensitivity corresponding to the polymerase chain reaction (PCR) but was implemented at a fixed temperature (39 °C) over 30 min. No unspecific reactions with Pectobacterium, Clavibacter, and other Dickeya species were observed. Detection of latent infection of D. solani in the potato tubers by the developed RPA-LFA was verified by PCR. The obtained results confirmed that RPA-LFA has great potential for highly sensitive detection of latent infection.

Lon Protease Is Important for Growth Under Stressful Conditions and Pathogenicity of the Phytopathogen, Bacterium Dickeya solani.

The Lon protein is a protease implicated in the virulence of many pathogenic bacteria, including some plant pathogens. However, little is known about the role of Lon in bacteria from genus Dickeya. This group of bacteria includes important potato pathogens, with the most aggressive species, D. solani. To determine the importance of Lon for pathogenicity and response to stress conditions of bacteria, we constructed a D. solani Δlon strain. The mutant bacteria showed increased sensitivity to certain stress conditions, in particular osmotic and high-temperature stresses. Furthermore, qPCR analysis showed an increased expression of the lon gene in D. solani under these conditions. The deletion of the lon gene resulted in decreased motility, lower activity of secreted pectinolytic enzymes and finally delayed onset of blackleg symptoms in the potato plants. In the Δlon cells, the altered levels of several proteins, including virulence factors and proteins associated with virulence, were detected by means of Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) analysis. These included components of the type III secretion system and proteins involved in bacterial motility. Our results indicate that Lon protease is important for D. solani to withstand stressful conditions and effectively invade the potato plant.