Relationship of Resistance-Related Enzyme activity and Salicylic Acid content in Phalaenopsis Species with different levels of resistance to DIckeya Dadantii

ABSTRACT Orchids (Phalaenopsis) are ornamental plants that are cultivated commercially and in great demand in the market. Soft-rot disease (SRD) caused by the necrotrophic pathogen Dickeya dadantii is a cause of considerable economic loss to cultivators of many orchid species. Our previous experiment identified a limited number of species that were resistant to D. dadantii. This study aimed to validate the resistance level of four Phalaenopsis species in a detached leaf inoculation protocol to identify the resistance mechanism(s) involved. Soft-rot symptom diameter was measured from 6 to 18 hours post-inoculation (HPI) with D. dadantii. Disease assessment confirmed that P. amboinensis is a resistant species, P. pantherina is a susceptible species, and P. amabilis and P. schilleriana are very susceptible species. There was no difference in the lignin content between the resistant and very susceptible species. Detailed observation of resistant and very susceptible species, P. amboinensis vs. P. amabilis, revealed higher phenylalanine ammonia-lyase (PAL) and peroxidase (POD) in P. amabilis than in P. amboinensis. In contrast, there was higher salicylic acid (SA) content in P. amboinensis than in P. amabilis. These results suggest that POD and PAL activities may not be effective in defense against soft-rot disease, while SA plays an important role in the resistance of P. amboinensis to D. dadantii. Low PAL activity in P. amboinensis implies that the SA contents from the isochorismate pathway may be involved in the mechanism of P. amboinensis resistance to D. dadantii. Therefore, endogenous SA content may be a good indicator for screening resistant species in Phalaenopsis.

Improving the Thermo-Activity and -Stability of Pectate Lyase from Dickeya dadantii DCE-01 for Ramie Degumming

To improve the thermal stability of pectate lyase for ramie degumming, we modified the novel pectate lyase gene (pelG403) derived from the Dickeya dadantii DCE-01 high-efficiency ramie degumming strain by site-directed mutagenesis. Twelve mutants were acquired, wherein a prospective mutant (A129V) showed better enzyme activity and thermal stability. Compared with the wild type (PelG403), the specific enzyme activity and the optimal reaction temperature of A129V in the fermentation broth increased by 20.1%, and 5 °C, respectively. Under the conditions of 55 °C and pH 9.0, the weightlessness rate of ramie raw materials of A129V increased by 6.26%. Therefore, this study successfully improved the enzyme activity and heat resistance of PelG403 in an alkaline environment, which may contribute to the development of enzyme preparations and the elucidation of the mechanism for ramie bio-degumming.

Complete Genome Sequence of Dickeya dadantii subsp. dieffenbachiae Strain S3-1, Isolated from a White-Flowered Calla Lily in Taiwan

Erwinia chrysanthemi S3-1 is a bacterial soft rot pathogen of the white-flowered calla lily. The complete genome sequence of the strain was determined and used to reclassify the strain as Dickeya dadantii subsp. dieffenbachiae . The sequence will be useful to study plant host-driven speciation in strains of D. dadantii .

Genome-wide identification of genes important for growth of Dickeya dadantii and D. dianthicola in potato (Solanum tuberosum) tubers

Dickeya species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (Solanum tuberosum). Using random barcode transposon-site sequencing (RB-TnSeq), we generated genome-wide mutant fitness profiles of Dickeya dadantii 3937, Dickeya dianthicola ME23, and Dickeya dianthicola 67-19 isolates collected after passage through several in vitro and in vivo conditions. Tubers from the potato cultivars “Atlantic”, “Dark Red Norland”, and “Upstate Abundance” provided highly similar conditions for bacterial growth. Using the homolog detection software PyParanoid, we matched fitness values for orthologous genes in the three bacterial strains. Direct comparison of fitness among the strains highlighted shared and variable traits important for growth. Bacterial growth in minimal medium required many metabolic traits that were also essential for competitive growth in planta, such as amino acid, carbohydrate, and nucleotide biosynthesis. Growth in tubers specifically required the pectin degradation gene kduD. Disruption in three putative DNA-binding proteins had strain-specific effects on competitive fitness in tubers. Though the Soft Rot Pectobacteriaceae can cause disease with little host specificity, it remains to be seen the extent to which strain-level variation impacts virulence.

Scaffolding protein GspB/OutB facilitates assembly of the Dickeya dadantii type 2 secretion system by anchoring the outer membrane secretin pore to the inner membrane and to the peptidoglycan cell wall.

The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal and human bacterial pathogens. This multi-protein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed as pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in Dickeya, the pilotin acts in concert with the scaffolding protein GspB. Deletion of gspB profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic Homology Region domain that interacts directly with the N-terminal secretin domain. Site-specific photo cross-linking unravels molecular details of the GspB-GspD complex in vivo. We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB scaffolds the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or a pilotin GspS.

Identification of new Dickeya dadantii virulence factors secreted by the type 2 secretion system

Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system named Out is necessary for bacterial virulence. Its study in D. dadantii showed that it secretes a wide range of pectinolytic enzymes. However, the full repertoire of exoproteins it can secrete has not been identified. No secretion signal present on the protein allows the identification of substrates of a type 2 secretion system. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced encoding a protein with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and showed that Svf proteins are present with a variable number of copies, up to three in D. fanghzongdai, in other Pectobacteriaceae. This work opens the way to the study of the role in virulence of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.

RNA Chaperones Hfq and ProQ Play a Key Role in the Virulence of the Plant Pathogenic Bacterium Dickeya dadantii

Dickeya dadantii is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the transcription of its genes encoding the main virulence functions, little information is available on the post-transcriptional regulation of these functions. We investigated the involvement of the RNA chaperones Hfq and ProQ in the production of the main D. dadantii virulence functions. Phenotypic assays on the hfq and proQ mutants showed that inactivation of hfq resulted in a growth defect, a modified capacity for biofilm formation and strongly reduced motility, and in the production of degradative extracellular enzymes (proteases, cellulase, and pectate lyases). Accordingly, the hfq mutant failed to cause soft rot on chicory leaves. The proQ mutant had reduced resistance to osmotic stress, reduced extracellular pectate lyase activity compared to the wild-type strain, and reduced virulence on chicory leaves. Most of the phenotypes of the hfq and proQ mutants were related to the low amounts of mRNA of the corresponding virulence factors. Complementation of the double mutant hfq-proQ by each individual protein and cross-complementation of each chaperone suggested that they might exert their effects via partially overlapping but different sets of targets. Overall, it clearly appeared that the two Hfq and ProQ RNA chaperones are important regulators of pathogenicity in D. dadantii. This underscores that virulence genes are regulated post-transcriptionally by non-coding RNAs.

Interplay between Coumarin Accumulation, Iron Deficiency and Plant Resistance to Dickeya spp.

Coumarins belong to a group of secondary metabolites well known for their high biological activities including antibacterial and antifungal properties. Recently, an important role of coumarins in plant resistance to pathogens and their release into the rhizosphere upon pathogen infection was discovered. It is also well documented that coumarins play a crucial role in the Arabidopsis thaliana growth under Fe-limited conditions. However, the mechanisms underlying interplay between plant resistance, accumulation of coumarins and Fe status, remain largely unknown. In this work, we investigated the effect of both mentioned factors on the disease severity using the model system of Arabidopsis/Dickeya spp. molecular interactions. We evaluated the disease symptoms in Arabidopsis plants, wild-type Col-0 and its mutants defective in coumarin accumulation, grown in hydroponic cultures with contrasting Fe regimes and in soil mixes. Under all tested conditions, Arabidopsis plants inoculated with Dickeya solani IFB0099 strain developed more severe disease symptoms compared to lines inoculated with Dickeya dadantii 3937. We also showed that the expression of genes encoding plant stress markers were strongly affected by D. solani IFB0099 infection. Interestingly, the response of plants to D. dadantii 3937 infection was genotype-dependent in Fe-deficient hydroponic solution.

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.