The plant pathogenic bacterium Pseudomonas savastanoi, the causal agent of olive and oleander knot disease, uses the so-called "indole-3-acetamide pathway" to convert tryptophan to indole-3-acetic acid (IAA) via a two-step pathway catalyzed by enzymes encoded by the genes in the iaaM/iaaH operon. Moreover, pathovar nerii of P. savastanoi is able to conjugate IAA to lysine to generate the less biologically active compound IAA-Lys via the enzyme IAA-lysine synthase encoded by the iaaL gene. Interestingly, iaaL is now known to be widespread in many Pseudomonas syringae pathovars, even in the absence of the iaaM and iaaH genes for IAA biosynthesis.Here, two knockout mutants, DiaaL and DiaaM, of strain Psn23 of P. savastanoi pv. nerii were produced. Pathogenicity tests using the host plant Nerium oleander showed that DiaaL and DiaaM were hypervirulent and hypovirulent, respectively and these features appeared to be related to their differential production of free IAA. Using the Phenotype Microarray approach, the chemical sensitivity of these mutants was shown to be comparable to that of wild-type Psn23. The main exception was 8 hydroxyquinoline, a toxic compound that is naturally present in plant exudates and is used as a biocide, which severely impaired the growth of DiaaL and DiaaM, as well as growth of the non-pathogenic mutant DhrpA, which lacks a functional Type Three Secretion System (TTSS). According to bioinformatics analysis of the Psn23 genome, a gene encoding a putative Multidrug and Toxic compound Extrusion (MATE) transporter, was found upstream of iaaL. Similarly to iaaL and iaaM, its expression appeared to be TTSS-dependent. Moreover, auxin-responsive elements were identified for the first time in the modular promoters of both the iaaL gene and the iaaM/iaaH operon of P. savastanoi, suggesting their IAA-inducible transcription. Gene expression analysis of several genes related to TTSS, IAA metabolism and drug resistance confirmed the presence of a concerted regulatory network in this phytopathogen among virulence, fitness and drug efflux.
Protection of plants against bacterial diseases still mainly relies on the use of chemical pesticides, which in Europe correspond essentially to copper-based compounds. However, recently plant diseases control is oriented towards a rational use of molecules and extracts, generally with natural origin, with lower intrinsic toxicity and a reduced negative environmental impact. In this work, polyphenolic extracts from vegetable no food/feed residues of typical Mediterranean crops, as Olea europaea, Cynara scolymus, and Vitis vinifera were obtained and their inhibitory activity on the Type Three Secretion System (TTSS) and the Quorum Sensing (QS) of the Gram-negative phytopathogenic bacterium Pseudomonas savastanoi pv. nerii strain Psn23 was assessed. Extract from green tea (Camellia sinensis) was used as a positive control. Collectively, the data obtained through gfp-promoter fusion system and real-time PCR show that all the polyphenolic extracts here studied have a high inhibitory activity on both the TTSS and QS of Psn23, without any depressing effect on bacterial viability. Extracts from green tea and grape seeds were shown to be the most active. Such activity was confirmed in planta by a strong reduction in the ability of Psn23 to develop hyperplastic galls on explants from adult oleander plants, as well as to elicit hypersensitive response on tobacco. By using a newly developed Congo red assay and an ELISA test, we demonstrated that the TTSS-targeted activity of these polyphenolic extracts also affects the TTSS pilus assembly. In consideration of the potential application of polyphenolic extracts in plant protection, the absence of any toxicity of these polyphenolic compounds was also assessed. A widely and evolutionary conserved molecular target such as Ca2+-ATPase, essential for the survival of any living organism, was used for the toxicity assessment.
A multiphasic approach was used to decipher the phenotypic features, genetic diversity, and phylogenetic position of 46 Curtobacterium spp. strains isolated from dry beans and other annual crops in Iran and Spain. Pathogenicity tests, resistance to arsenic compounds, plasmid profiling and BOX-PCR were performed on the strains. Multilocus sequence analysis (MLSA) was also performed on five housekeeping genes (i.e., atpD, gyrB, ppk, recA, and rpoB) of all the strains, as well as five pathotype strains of the species. Pathogenicity test showed that six out of 42 strains isolated in Iran were nonpathogenic on common bean. Despite no differences found between pathogenic and nonpathogenic strains in their plasmid profiling, the former were resistant to different concentrations of arsenic, while the latter were sensitive to the same concentrations. Strains pathogenic on common bean were polyphyletic with at least two evolutionary lineages (i.e., yellow-pigmented strains versus red/orange-pigmented strains). Nonpathogenic strains isolated from solanaceous vegetables were clustered within either the strains of C. flaccumfaciens pv. flaccumfaciens or different pathovars of the species. The results of MLSA and BOX-PCR analysis were similar to each other and both methods were able to discriminate the yellow-pigmented strains from the red/orange-pigmented strains. A comprehensive study of a worldwide collection representing all five pathovars as well as nonpathogenic strains of C. flaccumfaciens is warranted for a better understanding of the diversity within this phytopathogenic bacterium.
During the last years, many evidences have been accumulating about the phytohormone indole-3-acetic acid (IAA) as a multifaceted compound in the microbial world, with IAA playing a role as a bacterial intra and intercellular signaling molecule or as an effector during pathogenic or beneficial plant–bacteria interactions. However, pretty much nothing is known on the mechanisms that bacteria use to modulate IAA homeostasis, in particular on IAA active transport systems. Here, by an approach combining in silico three-dimensional (3D) structural modeling and docking, mutagenesis, quantitative gene expression analysis, and HPLC FLD auxin quantitative detection, for the first time a bacterial multidrug and toxic compound extrusion (MATE) transporter was demonstrated to be involved in the efflux of IAA, as well as of its conjugate IAA–Lysine, in the plant pathogenic hyperplastic bacterium Pseudomonas savastanoi pv. nerii strain Psn23. Furthermore, according to the role proved to be played by Psn23 MatE in the development of plant disease, and to the presence of Psn23 MatE homologs in all the genomospecies of the P. syringae complex, this membrane transporter could likely represent a promising target for the design of novel and selective anti-infective molecules for plant disease control.
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