Virulence Factors of Plant Growth-Promoting Bacteria: Analysis of Pseudomonas protegens Genomes

. Pseudomonas protegens is the species of plant growth-promoting bacteria, which is widely used in agriculture. In article, previously unknown virulence factors of this microorganism are revealed. When researching the three bacterial genomes P. protegens , genes encoding adherence, antiphagocytosis, iron uptake, biofilm formation, immune evasion, serum resistance, and other virulence traits have been found. More research is needed to learn the role of predicted virulence factors in phytopathology and medicine.


Introduction
Plant growth-promoting bacteria (PGPB) are a group of beneficial microorganisms that include 60 bacterial genera, such as Bacillus, Pseudomonas, and Burkholderia, which widely colonize plant leaves and soil, promote plant growth, and/or inhibit pathogen infection [1].One of them is the species Pseudomonas protegens, which was first described by Alban Ramette and coworkers in 2011 as widespread plant-protecting bacteria producing the antimicrobial compounds 2,4-diacetylphloroglucinol and pyoluteorin [2].
Known as biocontrol agents, these pseudomonads exhibit antibacterial and antifungal activity.It found a practical application in various fields of agriculture.For example, volatile organic compounds produced by acid-tolerant P. protegens CLP-6 had excellent inhibitory effects on Ralstonia solanacearum, which is the causative agent of tobacco bacterial wilt [3].The endophytic bacterium P. protegens NSJ-2101 inhibited the apple ring rot on postharvest fruits by activating the defense system of apple fruit and repressing the pathogenic factor of Botryosphaeria dothidea [4].Cell-free secretions from P. protegens PBL3 inhibited the growth of bacterium Burkholderia glumae in vitro and also prevented B. glumae from causing bacterial panicle blight of rice [5].Bacterial consortium of three Chilean strains of P. protegens inhibited wheat crown and root rot pathogens Gaeumannomyces graminis var.tritici, Rhizoctonia cerealis, and Fusarium culmorum [6].The strain of P. protegens Pf-5 strongly inhibited the oomycete pathogen Aphanomyces euteiches (pea Aphanomyces root rot causative agent) [7].Application of the bacterial strain P. protegens DA1.2 increased the amount of harvested bread wheat (Triticum aestivum L.) grain from 2.0-2.2t/ha to 3.2-3.6t/ha [8].
As per current information, infection caused by P. protegens in plant or animal/human host has not been described.However, virulence factors of P. protegens have not been analyzed.In this article the present author analyzes the genomes of different P. protegens strains to search for a bacterial potential pathogenic attributes.

Materials and methods
The complete genomes of three P. protegens strains were analyzed (Table 1).The type strain CHA0 was isolated from the roots of tobacco in Swiss soil, which was naturally suppressive to black root rot in tobacco caused by Thielaviopsis basicola [9].P. protegens SN15-2 was isolated from the rhizosphere of tomato roots in Shanghai, China [10].The biocontrol strain P. protegens Cab57 was isolated from the rhizosphere of shepherd's purse growing in a field in Hokkaido, Japan, by screening the antibiotic producers [11].For comparison, complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen, was used [12].The characterization of genomes was carried out using the Virulence Factor Database (VFDB, http://www.mgc.ac.cn/VFs/), which is an integrated and comprehensive online resource for curating information about virulence factors of bacterial pathogens.Since its inception in 2004, VFDB has been dedicated to providing up-to-date knowledge of VFs from various medically significant bacterial pathogens.Instead of using simple BLAST searches, VFanalyzer first constructs orthologous groups within the query genome and preanalyzed reference genomes from VFDB to avoid potential false positives due to paralogs.Then, it conducts iterative and exhaustive sequence similarity searches among the hierarchical pre-build datasets of VFDB to accurately identify potential untypical/strainspecific VFs.Finally, via a context-based data refinement process for VFs encoded by gene clusters, VFanalyzer can achieve relatively high specificity and sensitivity without manual curation [13].

Results and Discussion
The results of investigation showed, that although highlighted strains were isolated in different parts of the world (two in Asia, and one in Europe), and from the rhizosphere of miscellaneous plants, all of them found virulence factors.These factors are grouped into several categories (Table 2).First step in bacterial pathogenesis is usually adherence to tissue.Bacterial motility is an important capability for a successful pathogen to avoid hostile environments and discover useful resources for survival.Flagella contributes to swimming motility, play a role in biofilm formation, which largely improves their resistance to antimicrobials and host immunities to contribute to the survival, dispersion and colonization of the bacteria.By type IV pili bacterial cells attach to host cells, causing a twitching motility that allows the bacteria to move along the cell surface [13][14][15].Mucoid exopolysaccharide alginate also affects P. aeruginosa biofilm development and architecture [16].
Iron is an essential nutrient for the proliferation and pathogenicity of bacterial pathogens.The well-characterized host defense strategy of iron sequestration highlights the crucial role of iron acquisition systems in bacterial pathogenesis [13].Pyoverdine is effective at acquiring iron from transferrin and lactoferrin [17].Pyochelin, a structurally unique siderophore possessing phenolate, but neither a hydroxamate nor a catecholate moiety, is effective at promoting iron in P. aeruginosa.Also, pyochelin also binds other transition metals (e.g.Mo(IV), Ni(II) and Co(II)) with appreciable affinity and is, in fact, implicated in the delivery of both Co(II) and Mo(IV) to P. aeruginosa cells [18].
Catalase (katA) detoxifies H2O2, protects against reactive oxygen species, a family of chemical that are oxidized version of molecular oxygen (hydrogen peroxide, superoxide, and hydroxyl radicals).Catalase:peroxidase (katG) degrades H2O2 and organic peroxides, the major role is to catabolize the peroxides generated by phagocyte.

Conclusion
The obtained results indicate that the beneficial saprophytic rhizosphere bacteria have virulence genes in their genome.Under certain conditions (for example, climate change, exposure to pesticides, or introduction into the immunodeficient organism) this circumstance is bound to be of great importance.

Table 1 .
Analyzed sequences of P. protegens strains in this work.

Table 2 .
Predicted virulence factors of P. protegens strains in comparison with P. aeruginosa PAO1.