- Original article
- Open Access
A genetic tool for production of GFP-expressing Rhodopseudomonas palustris for visualization of bacterial colonization
AMB Express volume 9, Article number: 141 (2019)
Development of a genetic tool for visualization of photosynthetic bacteria (PSB) is essential for understanding microbial function during their interaction with plant and microflora. In this study, Rhodopseudomonas palustris GJ-22-gfp harboring the vector pBBR1-pckAPT-gfp was constructed using an electroporation transformation method and was used for dynamic tracing of bacteria in plants. The results showed that strain GJ-22-gfp was stable and did not affect the biocontrol function, and the Confocal Laser Scanning Microscopy (CLSM) results indicated it could successfully colonised on the surface of leaf and root of tobacco and rice. In tobacco leaves, cells formed aggregates on the mesophyll epidermal cells. While in rice, no aggregate was found. Instead, the fluorescent cells colonise the longitudinal intercellular spaces between epidermal cells. In addition, the results of strain GJ-22 on the growth promotion and disease resistance of tobacco and rice indicated that the different colonization patterns might be related to the bacteria could induce systemic resistance in tobacco.
Rhodopseudomonas palustris is a Gram-negative non-sulfur purple bacterium that can propagate through budding and can grow under anaerobic condition in the light. To date, R. palustris is widely used in the aquaculture industry, hazardous material degradation, wastewater treatment, livestock and poultry production, and environment protection (Wang et al. 2008; Deyong et al. 2005; Tao et al. 2013; Li et al. 2005). A previous study has also demonstrated that R. palustris can be used to improve soil fertility, plant growth, and plant disease resistance (Su et al. 2017). R. palustris is considered to be beneficial for sustainable agriculture because it is non-toxic to humans and animals and does not produce toxic residues in soil and water. In addition, R. palustris and other photosynthetic bacteria can be used to promote degradation of pesticides and removal of heavy metals from soil during crop production (He 2007).
The activities of bacteria on their host plants and the mechanism of how to promote plant growth and disease resistance are poorly understood, mainly due to lack of powerful and valuable tools to monitor their colonization during the infection cycle in plants. Development of a genetic tool for visualization is essential for understanding the microbial function during their interaction with plants and microflora. Many reports have suggested using specific bacterial antibiotic resistance as markers to track the survival and colonization pattern of R. palustris strains in plants and animals (Aarts et al. 2001; Flint et al. 1989). Although the methods using antibiotic markers are simple, rapid and low cost, the antibiotic markers are unstable and easy to lose during the study (Nairn and Chanway 2002; Torres et al. 2013). Also, these methods could not exclude the effects generated by contaminated bacterial strains.
To establish a powerful and valuable tool for the study of R. palustris, a green fluorescent protein (gfp) gene was introduced into the genome of R. palustris to track the bacterial dynamic colonization in plants. Using gfp as a marker to investigate protein function in cells or to track pathogen spread in plants or animals has attracted considerable attentions since the gene was cloned from Aequorea victoria (Prasher et al. 1992; Niedenthal et al. 1996; Inouye and Tsuji 1994). In 1997, Tombolini et al. (1997) constructed a gfp expression cassette tagged Pseudomonas bacteria to monitor the colonization, spatial distribution and survival of these microorganisms. Bloemberg et al. (1997) constructed plasmids which expressed a bright mutant of gfp in Pseudomonas spp. strain WCS365 to demonstrate the association of bacteria with tomato seedling roots. Chen et al. (2005) used gfp as a molecular marker to monitor Bacillus brevis survival in soil. In 2016, Teh et al. (2016) engineered a gfp-tagged bacterium E. munditi to observe its colonization in S. littoralis. These studies indicated that gfp can be used as a useful and stable marker for visualization of bacterial colonization. However, due to lack suitable vectors, the construction of gfp-tagged R. palustris has not been successful. In this study, the gfp-tagged strain GJ-22-gfp along with the gene promoter and terminator of R. palustris was constructed using an electroporation transformation method. Expression of gfp in this transgenic GJ-22 strain was stable and did not affect the biocontrol function. CLSM results indicated R. palustris GJ-22 gives a different colonise pattern on the surface of leaves and roots in tobacco and rice, and the results of later experiments indicated that this might due to the strains induce plants to produce induced resistance. In a word, the construction of gfp-labeled R. palustris would greatly facilitate the understanding of the interaction of plant–microbial, which will be of historic significance for the future study.
Materials and methods
Bacterial strains, culture conditions and plasmids
The bacterial strains and plasmids used in this experiment are listed in Additional file 1: Table S1. R. palustris GJ-22 strain (CGMCC: 17356) were grown in the medium followed Song (2007) with minor modification, named PSB medium. The medium comprised: 0.5 g [NH4]2SO4, 0.5 g NaAc, 1 g K2HPO4, 0.05 g FeSO4, 0.05 g H3BO3, 0.05 g Na2MoO4 and 1.5 g yeast extract dissolved in 1-l of distilled water. The growth medium was adjusted to pH 7.3 then autoclaved for 50 min at 121 °C. R. palustris GJ-22 were grown in anaerobic condition under light without antibiotics. Escherichia coli were cultured in Luria Bertani (LB) medium at 37 °C with shaking at 200 rpm overnight (Deininger 1989). Magnaporthe oryzae strain 70-15 was obtained from the Fungal Genetics Stock Centre, Kansas City, KS, USA. The strain was maintained on CM medium (Talbot et al. 1993). Agar was also added to medium to reach 1.5 g L−1 when sub-cultured. Kanamycin (50 mg mL−1) or ampicillin (100 mg mL−1) was also added to the medium when required. Strain GJ-22 was cultured anaerobically in a two-layer medium plate with 1.3% agarose in the lower layer and 1.8% agarose in the top layer.
Construction of strain GJ-22-gfp
Rhodopseudomonas palustris GJ-22 strain was inoculated at a rate of 1% inside a flask and kept at 30 °C for 3 days under 7500 lx light. After genomic DNA of R. palustris GJ-22 was extracted using the CTAB method with OD660 at 1.0 (Doyle 1991), the resulting genomic DNA was checked in 1% agarose gels through electrophoreses. Promoter (pckA-P) and terminator (pckA-T) of Phosphoenolpyruvate carboxykinase (pckA) gene (Inui et al. 1999) were PCR amplified from the DNA of R. palustris GJ-22 using primers pckA-P F/R and pckA-T F/R (primers were show on Additional file 1: Table S2, and the sequences of pckA were show on Additional file 2). The primer of pckA-P F and pckA-T R were digested with Kpn I and Bgl II restriction enzymes respectively, and the primer pckA-T F and pckA-T R were digested with Bgl II and EcoR I restriction enzymes respectively. These two digested PCR products were first ligated together at the Bgl II site and then inserted into the pBBR1MCS-2 vector (GenBank sequence: U23751) at the pre-digested Kpn I and EcoR I sites to generate pBBR1-pckAPT (Fig. 1). This vector was propagated in the Trans1-T1 competent cells (TransGen Biotech technology, Beijing, China) and plasmid isolation using a plasmid extraction kit (TransGenBiotech, BeiJing, China).
A green fluorescent protein (gfp) gene (GeneBank: KP294375.1) was PCR amplified from vector pRK415 with primer gfpmut3 F/R (primers were show on Additional file 1: Table S2, and the sequences of gfp were show on Additional file 2). The PCR products were purified (OMEGA Bio-tek, USA), then cloned into the pEasy-T1 vector, resulting vector (pEasy-T1-gfp) was transformed into the Trans1-T1 cells for propagation. The gfp gene fragment was released from the pEasy-T1-gfp vector through overnight Bgl II enzyme digestion at 37 °C, isolated using a gel extraction kit (OMEGA Bio-tek, USA), and then inserted between the pckA promoter (pckA-P) and pckA terminator (pckA-T) of vector pBBR1-pckAPT to generate pBBR1-pckAPT-gfp (sequence of pBBR1-pckAPT-gfp was shown in Additional file 3). The integrity of the pBBR1-pckAPT-gfp vector was sequenced by the TsingKe Biological Technology (ChangSha, China). This vector was further propagated in E. coli BL21 (DE3) cells (TransGen Biotech technology, Beijing, China) for gfp expression.
GFP expression in E. coli BL21 (DE3) cells were induced by addition of isopropyi-d-thiogalactopyranoside (IPTG) into the culture medium as previously described (Lewis and Marston 1999). The gfp expression of IPTG-treated cells was examined under a confocal fluorescent microscope with 488 nm excitation and collecting fluorescence in the range 500–550 nm (Nikon C2 plus, Nikon, Japan). The cells were photographed with Nikon CFI Plan Apochromat VC 60XH, NA:1.4, WD: 0.13 mm. Competent R. palustris GJ-22 cells were prepared as previously described (Pelletier et al. 2008). Briefly, R. palustris GJ-22 culture was grown under anaerobic condition till the OD660 at 0.4, and then cells were pelleted by centrifugation at 13,751g for 10 min at 4 °C. The pellet cells were rinsed three times with ice-cold sterile water and then resuscitated in a 10% glycerol solution. Finally, cells were stored in 1.5 mL sterile centrifuge tubes at − 80 °C immediately. Before transformation, the frozen R. palustris cells (50 µL) were thawed on ice for 20 min and mixed with 5 ng of pBBR1-pckAPT-gfp DNA and 80 μL of sterile ice-cold distilled water inside a 2 mm Gene Pulser cuvette. After 10 min incubation on ice, electroporation was performed using the Eppendorf Eporator (Eppendorf North America, Hauppauge, NY, USA) set at 2.5 kV voltage, 25 µF capacitance, and 100 Ω resistance. After electroporation, the cells were incubated 2 min at room temperature, then grown in 10 mL culture medium for 20 h at 30 °C under light. Positive transformants were selected on agar medium containing kanamycin (50 mg mL−1) and cultured for 4 days at 30 °C under light.
Quantification of gfp expression and stability of strain GJ-22-gfp
GJ-22-gfp cells were determined using a confocal fluorescent microscope with 488 nm excitation and collecting fluorescence in the range 500–550 nm (Nikon C2 plus, Nikon, Japan). Images of the cells were captured using a Nikon CFI Plan Apochromat VC 60XH (Nikon, Japan) and processed using the Photoshop 7.0 software for stitching.
Stability of strain GJ-22-gfp was tested by growing the cultures through 15 consecutive passages on the solid culture medium with and without kanamycin. Briefly, for each passage, the GJ-22-gfp cells were grown at 30 °C for 4 days in the liquid medium without antibiotics, and then the cells were inoculated to agar medium plates with and without kanamycin. Colonies on each plate were calculated and then examined under a fluorescence microscope (Olympus BX51, Japan). This experiment was repeated three times.
The growth curves of wild type and gfp-labeled strain were measured to determine whether the addition of gfp affects the growth metabolism. Wild type strain GJ-22 and GJ-22-gfp were cultured in the same condition. The cells were centrifuged at 8000g and resuscitated with sterile liquid medium when the cells were grown at OD660 1.0. The concentration of cells was adjusted to OD660 at 1.0. Diluted cells were inoculated to fresh liquid medium and grown at 30 °C with 7500 lx. The cell cultures were measured for cell concentrations at 12 h post culturing and then once every 12 h till 108 h to generate the cell growth curves. This experiment was repeated three times.
To determine the plasmid stability in GJ-22-gfp during the plant surface colonization and effect of plasmid on strain’s ability for plant surface colonization, bacterial cells were sampled with the following method at the indicated post-inoculation times. Five-week-old tobacco and rice were inoculated using the method of foliar spray and root irrigation with 10 mL wild type strain GJ-22 and GJ-22-gfp (5 × 107 CFU mL−1). The third and fourth leaves (down from the top) and roots (5 cm) of tobacco and rice were harvested from individual assayed plants and pooled at 0th, 24th, 48th, 72th h. Five gram tissues were sampled from each harvested sample and submerged in 100 mL PBS conical flask for 20 min. Samples were then sonicated for 10 min at 47 kHz with 150 rpm shaking. Bacterial cells were pelleted from each sample by 10 min centrifugation at 10,000g. The pelleted cells were resuscitated with 2 mL PBS buffer and remove the remaining plant debris by 12,000g centrifugation for 10 min.
The bacterial population density of GJ-22 and GJ-22-gfp in phyllosphere and rhizosphere of tobacco and rice were determined by plating the serially cell samplings on the PSB agar medium. The serially diluted GJ-22 cells were platted on the PSB agar medium without kanamycin, and the serially diluted cells of strain GJ-22-gfp were platted on the PSB agar medium with and without kanamycin respectively. Plates were subjected to culture for 5 days before the colony-forming units (CFU) on each plate were calculated.
Plant growth conditions and colonisation visualization
Seeds of tobacco cv. (Nicotiana benthamiana cv. Changsha, collected from the experimental research station) and rice seeds (Longping seeds, Changsha) were obtained from horticultural crop pest control laboratory of Hunan, and these seeds were sterilized with 75% ethanol for 5 min, then rinsed with sterile water thoroughly. Four treatments were set in this experiment: GJ-22 suspension, GJ-22-gfp suspension, PSB medium and ddH2O were as the control blank (CK). To observe whether the insertion of exogenous gene would influence the interaction effect between bacteria and plant, the root length and dry mass of tobacco and rice were examined. The treatments was immersed in seeds for 24 h, and the seeds were placed in a wet filter paper culture dish and germinated at 25 °C under red light. Three dishes of each treatment and 100 seeds of each dish were cultured with liquid after germination. Root length and dry mass of tobacco and rice were measured on the 14th and 21th day, respectively.
For visualization the colonization dynamics, 5-week-old tobacco and rice were root-irrigated with R. palustris GJ-22-gfp (5 × 107 CFU mL−1) by pouring 15 mL of R. palustris GJ-22-gfp culture into each pot. In a separate experiment, the same volumes of R. palustris GJ-22-gfp were sprayed onto leaves of plants. The equal volumes of medium were used as the control. At 24th, 48th and 72nd day post inoculation (dpi), the assayed plants were harvested, rinsed with sterile phosphate buffered saline (PBS) solution, and the fresh leaves and roots were collected to examine the R. palustris GJ-22-gfp colonisation under the confocal microscope. Images were captured under the confocal microscope equipped with a 20× objective (N.A–0.75) and a 488 nm emission and collecting fluorescence in the range 500–550 nm.
Detection of plants growth promotion
Based on the results of CLSM, it is hypothesized that the GJ-22 could formed bacterial aggregates in tobacco leaves, which could possess ISR-inducing properties. Nicotiana benthamiana (TMV systematic host) and rice seeding were cultured 7 days under red light, and then transplanted into single pot. After cultured 7 days, the plants with the same growth condition were selected to divide into four groups, and 30 plants were used for each treatment. These treatments were as following: (i) Spraying the leaves with 5 mL of bacterial suspension (108 CFU mL−1); (ii) root irrigation with 5 mL bacterial suspension (108 CFU mL−1); (iii) treat plants with the same volume of medium (foliar spray and root irrigation); (iv) treatment plants with the same volume of ddH2O as the blank control. The leaf treatment was foliar spray to shoot of tobacco and rice to soaring wet. Dry mass of tobacco and rice were quantified at 1st, 2nd, 3rd and 7th day after disposed. The dry mass of each plant were weighted after drying at 60 °C for 3 days. This experiment was repeated three times.
TMV and Magnaporthe oryzae determination
TMV accumulation was tested by enzyme-linked immunosorbent assay (ELISA), the treatment was according the manufacturer instruction (IBL-America, Minneapolis, USA). And the blast lesions of M. oryzae were counted in rice leaves. Tobacco and rice were pre-treated with the same treatment as plants growth promotion: foliar spray to shoot, root irrigation, medium and ddH2O respectively after cultured 4 weeks. TMV particles and M. oryzae were inculcated the next day of treatment. The samplings were collected at the time of 1st, 2nd, 3rd and 7th day after the tobacco inoculated with 20 µL TMV particles (10−5 mg mL−1) and the rice inoculated with 5 mL of M. oryzae (4.0 × 105 CFU mL−1). For tobacco, 1 g fresh leaf tissue of inoculated top blade from each different treatments and different times were collected, flash-frozen and grind with 1 mL of PBS buffer (pH = 7.4), then centrifuged with 8801g for 20 min. The suspensions were detected by ELISA, and the TMV accumulation was determined with a microplate spectrophotometer at 450 nm. For rice, samplings were obtained to count the blast lesions of M. oryzae of in per unit area.
All the experiments performed in this study were repeated three times. Bacterial population data were log transformed before subjected for further analysis. Statistical differences between treatments were determined by the Tukey’s test using the software of SPSS statistics 17.0 (IBM Corp., New York, USA).
Construction of gfp-labeled strain
The inserted pckA-gfp gene was confirmed through sequencing and gel detection, result showed a bright and single band at 750 bp. Expression of gfp in E. coli showed the strong green fluorescence. The positive GJ-22-gfp colonies were examined under the confocal fluorescence microscope. Results showed that almost all of transformed R. palustris GJ-22 cells showed a strong green fluorescence signal (Additional file 1).
To detection the stability of gfp-labed strain, strain GJ-22-gfp were cultured and examined for gfp expression through 15 passages in the culture medium with and without kanamycin. The result showed that about 10% of the colonies lost their gfp signal after five passages, and the stability rate was maintained at 79% after nine passages (Fig. 2a).
Growth curves of the wild type GJ-22 and transgenic GJ-22-gfp were determined by growing them in liquid medium without kanamycin. Results showed that the wild type and transgenic R. palustris GJ-22 grew similarly in the liquid medium without kanamycin. However, with the addition of kanamycin, the growth of GJ-22-gfp cells was lagged (Fig. 2b). This result indicated that insertion of pBBR1-pckAPT-gfp into R. palustris GJ-22 did not affect the growth, but with kanamycin, the growth of GJ-22-gfp was lagged. In identifying the biocontrol function of labeled strains, results showed that strain GJ-22-gfp could increase the root length and dry mass of rice and tobacco as wild type strain GJ-22, both higher than the blank control significantly, and no significant differences (P < 0.05) between the strain GJ-22-gfp and the wild type strain GJ-22 (Fig. 3A, B).
To determine the plasmid stability during plant surface colonization, the tagged-strain cells were washed off from plant surfaces at multiple time-points and cultured on plate medium with and without kanamycin. The results showed that there were no significant differences between the population sizes from the two plates, indicating the plasmid functioned stably during the plant surface colonization of the strain GJ-22-gfp (Fig. 3C–F).
The effect of plasmid on the strain´s ability to colonise plant surface as determined by comparing the population size between the wild type strain and tagged strain. During the bacterial colonization on plant surface, there was no significantly different population size detected between the two strain, indicating that the plasmid had no detectable influence on strain’s ability for plant surface colonization (Fig. 3C–F).
CLSM of visualization the colonization of GJ-22-gfp in tobacco and rice
The leaves and roots of tobacco and rice were sampled at 24th, 48th and 72nd h after inoculated with GJ-22-gfp. The leaves and root tips of the plants were collected to visualize the expression of strain GJ-22-gfp. The results of CLSM in tobacco and rice were shown in Figs. 4, 5. On the 24th h of inoculation, a large number of bacteria cells were observed to be unevenly accumulated on the surface of tobacco mesophyll cells without any special localization (Fig. 4a). On the 48th h, bacterial cells were adhesion on the epidermal cell groove of tobacco mesophyll cells. However, the complete colonization of the cells were not visualized (Fig. 4b). On the 72nd h sampling detection, numerous single cells formed aggregations and clumps of cells were observed to colonise on the epidermal cells and deep grooves between epidermal cells of leaves of tobacco, and the green fluorescent cells were seen filled with the stomata occasionally (Fig. 4c). And the uninoculated leaves showed no fluorescence cells (Fig. 4d). While on roots, numerous GJ-22 cells were observed on the surfaces of primary roots at the 24th h (Fig. 4e). The distribution of GJ-22-gfp cells on the root surface was random. The cells on roots were less than that of the leaf explicitly. On the 48th h, in addition to individual GJ-22-gfp cells, long strings of green fluorescent cells were also observed along the root epidermal cells (Fig. 4f). Root hairs were seen no GJ-22-gfp cells. While on the final sampling detection, GJ-22-gfp cells were observed on the root surface and occupied between longitudinal intercellular spaces of roots surface (Fig. 4g). No green fluorescent cells were seen in control roots of tobacco (Fig. 4h).
In rice, the CLSM results were shown in Fig. 5. Unlike tobacco, there was no cell aggregation on leaves and roots, high background fluorescence was easily visualized on rice. On the 24th h sampling detection, numerous single GJ-22-gfp cells were lined up along the leaf surface layer of rice (Fig. 5a). However, the results of 48th h sampling showed the bacterial cells on the leaves of rice to be decreased sharply, and the bacterial cells were observed to be in the linear distribution in the leaves damaged area throughout the entire length (Fig. 5b). On the 72nd h, there were only single cells occupied linearly between the longitudinal intercellular spaces of mesophyll epidermal cells (Fig. 5c). And there were no fluorescence cells observed on control treatment (Fig. 5d). On the root of rice, numerous fluorescence cells were seen adhesion on the root apex and root cap of the plant on the 24 h (Fig. 5e). While on the 48 h, most of the cells do not appeared in the apical and coronal of rice root, but found to be distributed linearly in the root extension (Fig. 5f). And the results of the last sampling also showed that most fluorescence cells were distributed on the root extension of rice, and the number of bacteria was reduced significantly (Fig. 5g). Similarly, no fluorescence cells were found on the rice roots of the uninoculated control (Fig. 5h).
Plant growth promotion and disease resistance test
The dry mass of tobacco (Fig. 6A) and rice (Fig. 6C) treated with the GJ-22-gfp shoot, root, medium and ddH2O were weighed. In tobacco, the shoot with GJ-22-gfp suspension were significantly higher than the other treatments from the 2nd days (P < 0.05) (Fig. 6A). In rice, on the 1st and 2nd day of treatment, the treatments with shoot and root were higher than blank control, but no significant (P > 0.05). On the 3rd day, the treatment with shoot and root were higher than medium and ddH2O significantly (P < 0.05), and on the sampling of 7th day, the shoot treatment was higher than root and blank control (Fig. 6C).
For the TMV accumulation assay, from the Fig. 6B, with the time over, the TMV accumulation in four treatments increased significantly. On the 1st day of inoculation, the TMV accumulation of shoot treated was higher than other treatments, but on the 2nd day, there had a tendency of the TMV accumulation was lower than root treated and blank control significantly (P < 0.05), and the TMV concentration of root treated was no significantly lower than blank control. While on detection on rice, the GJ-22 treated shoots and roots had no differences with blank control on the 1st and 2nd day. On the 3rd day of inoculation, there has a tendency of reduce the disease plaques, and samplings from 7th day, the lesion number of roots and shoots were less than treated with medium and ddH2O significantly (Fig. 6D).
Nowadays, gfp is frequently used in biological and biotechnological studies especially as a marker gene (Errampalli et al. 1999; Bloemberg 2007). Koch and co-workers (2001) developed a Tn7-GFP system to express gfp in strain Pseudomonas for the detection of bacteria cells in the barley rhizosphere. Xiong et al. (2013) produced a GFP-expressing Rhodococcus sp. D310-1 strain to study the stability of bacterium in soil and its ability to remove chlorimuron-ethyl from contaminated soils. In 2010, Lagendijk et al. (2010) developed a tool for the transformation of Gram-negative bacteria with a mCherry gene and used this fluorescence protein as a marker to visualize bacterial communities in biofilms formed on glass and tomato roots. However, due to the lack of appropriate vectors, no expression vector suitable for expressing foreign genes into R. palustris. As a plant growth promoting bacteria, R. palustris can not only enhance plant performance under various environment stresses through regulating different plant defense signaling pathways, but also inhibit pathogen infections (Pleban et al. 1995; Shishido et al. 2010; Delany et al. 2001; Nunkaew et al. 2014). To elucidate the mechanism controlling R. palustris colonization in plant and to establish a better field application strategy, knowledge and understanding related to R. palustris colonization process in its host plant is crucial.
In this study, a broad host range vector pBBR1MCS-2 was used as the basic carrier. A conserved promoter (pckA-P), terminator (pckA-T), and a ribosome binding site (AGGAGG) were added to the basic vector to produce the recombinant vector pBBR1-pckAPT-gfp. Before this study, three expression vectors (e.g., PVLT33-GFP, pBBR1MCS-2-pAMP-EGFP and pGreenpuro) were constructed and inserted into R. palustris GJ-22 cells, but no fluorescence signal was observed, then the fourth broad host range expression vector pBBR1MCS-2 was constructed based on the earlier reports (Park et al. 2010; Chin A Woeng et al. 2001). Therefore, a suitable promoter is crucial for any expression vector and can decide the efficiency of gene expression.
During the detection of strain stability, 10% of the gfp-labeled strain lost fluorescence after five passages, and the maintenance had fallen to 79% after nine passages. The reason for the disappearance of the fluorescence was still unclear, it was speculated that it is may be a result of the invisible modification and sharing of the foreign gene by the bacteria, which may caused by mismatching or due to lost the gfp gene during plasmid replication. In the determination of growth curves of labeled strain and wild-type strain, there was no significant difference in growth, but with the addition of antibiotics, there is a lag phenomenon in the growth stage, which may be the addition of kanamycin that put pressure on the host bacteria metabolism.
Rhodopseudomonas palustris GJ-22, in both cases for the gfp-labeled and the wild type strain, promoted the growth of tobacco and rice, and the effect was related to the hormones secreted by the bacteria (Su et al. 2017; Madhaiyan et al. 2006).
During the colonisation detection, the strain GJ-22 was observed to form aggregates colonised between the mesophyll cells and grooves in tobacco leaves, while in rice, the cells were not formed as the bacterial community as tobacco, but colonised on the longitudinal groove space of epidermal cells. The difference in colonization patterns between tobacco and rice may be attributed to the physiology differences between dicotyledonous and monocotyledonous plant. The differences in colonisation were also reported in methylobacter (Omer et al. 2004; Poonguzhali et al. 2008). It most likely that there were more epidermal cell grooves on the tobacco leaf, as there sites could provide more residual water and variable nutrients to microorganism, similar results were described various of availability compounds, such as, phenol (Sandhu et al. 2007), water (Axtell and Beattie 2002) or the distribution of fructose (Remus-Emsermann et al. 2011). Therefore, there are many different microhabitats offered by leaves. Remus-Emsermann et al. (2014) reported a surprising dense population of 104–105 bacteria mm−2 in leaf surface, colonised a wide range of microorganisms, including bacteria, fungi and oomycetes (Agler et al. 2016). And in the other words, the leaf surfaces are the better home to diverse bacterial communities (Remus-Emsermann and Schlechter 2018). The observations by CLSM showed the cells were distributed along the primary roots of tobacco and rice. From the results of CLSM, the population of cells in leaf was far greater than in root, and the number in tobacco was far greater than rice. The dominant colonization location of the bacterial cells could be related to the chemical compounds secreted by bacteria on the leaf surface and the anatomical difference of dicotyledonous plant (Gotz et al. 2006). In addition, the independent treated roots, leaves and stems of tobacco were visualized by CLSM, no fluorescence cells were observed; similarly, the strain GJ-22-gfp was not found in the stems and roots of the leaves independent treated leaves in tobacco. This indicated that the strain GJ-22 could not migrate from the root to the leaf and could not shuttle from the leaf to the root, either. The onset of induced systemic resistance usually achieved in plant growth-promoting bacteria (PGPB), and ISR will be a potential pathway for plant development (Van Loon 2007). The section of ISR induced strains have been greatly promoted worldwide (Wang et al. 2014; Berg 2015), but the difficulties in gfp-tagged photosynthetic bacteria leads to the shortage of biocontrol mechanism. Bacterial aggregates were formed on the mesophyll cells and grooves in tobacco leaves, and showed great growth and disease resistance characteristics, so we hypothesized that strain GJ-22 had the characteristics of inducing plants to produce ISR, which still needs to be verified by a large of experiments. On the other hand, the diversity changes of agricultural ecological environment also brought great changes to the stability of gfp-labeled strain, then the selection of suitable strains to adapt the environment is has a great significance for the development of biocontrol agents.
Generally, the tools of gfp-tagged R. palustris have always been a problem for many researchers. In this study, the gfp-labeled strain GJ-22-gfp was constructed successfully and was used for the tracking of R. palustris in the plant, which would help to understand the persistence of R. palustris in host plants, environment and its contribution to disease resistance and plant growth.
Availability of data and materials
The strains were available upon request. All data obtained have been included into the manuscript.
phosphate buffered saline
green fluorescent protein
- R. palustris :
Confocal Laser Scanning Microscopy
Agarose Gel Electrophoresis
Aarts HJM, Boumedine KS, Nesme X, Cloeckaert A (2001) Molecular tools for the characterisation of antibiotic-resistant bacteria. Vet Res 32:363–380. https://doi.org/10.1051/vetres:2001130
Agler MT, Ruhe J, Kroll S, Morhenn C, Kim S-T, Weigel D, Kemen EM (2016) Microbial hub taxa link host and abiotic factors to plant microbiome variation. PLoS Biol 14:e1002352. https://doi.org/10.1371/journal.pbio.1002352
Axtell CA, Beattie GA (2002) Construction and characterization of a proU-gfp transcriptional fusion that measures water availability in a microbial habitat. Appl Environ Microbiol 68:4604–4612. https://doi.org/10.1128/AEM.68.9.4604-4612.2002
Berg G, Erlacher A, Grube M (2015) The edible plant microbiome: importance and health issues. Principles of Plant-Microbe Interactions. Springer International Publishing. https://doi.org/10.1007/978-3-319-08575-3_44
Bloemberg GV (2007) Microscopic analysis of plant–bacterium interactions using auto fluorescent proteins. Eur J Plant Pathol 119(3):301–309. https://doi.org/10.1007/s10658-007-9171-3
Bloemberg GV, O’Toole GA, Lugtenberg BJ, Kolter R (1997) Green fluorescent protein as a marker for Pseudomonas spp. Appl Environ Microbiol 63(11):4543. https://doi.org/10.1002/(SICI)1097-0290(19971105)56:3%3c345:AID-BIT13%3e3.0.CO
Chen Y, Shen D, Yang M (2005) Use of green fluorescent protein as molecular marker for tagging Bacillus brevis, in soil under the control of a novel constitutive promoter F1. Folia Microbiol 50(5):437–442. https://doi.org/10.1007/BF02931426
Chin A Woeng TFC, Van den Broek D, De Voer G, Van der Drift KM, Tuinman S, Thomas-Oates JE, Lugtenberg BJJ, Bloemberg GV (2001) Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis pcl1391 is regulated by multiple factors secreted into the growth medium. Mol Plant Microbe Interact 14(8):969–979. https://doi.org/10.1094/MPMI.2001.14.8.969
Deininger P (1989) Molecular cloning: a laboratory manual. In: Sambrook J, Fritsch EF, Maniatis T (eds) Anal Biochem, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, vol 186, no 1, pp 182–183. http://doi.org/10.1016/0003-2697(90)90595-Z
Delany IR, Walsh UF, Ross I, Fenton AM, Corkery DM, O´Gara F (2001) Enhancing the biocontrol efficacy of Pseudomonas fluorescens F113 by altering the regulation and production of 2, 4-diacetylphloroglucinol. Plant Soil 232(1/2):195–205. https://doi.org/10.1023/a:1010306724455
Deyong Z, Xinqiu T, Xiangwen L, Chunhui Z, Yuanhua L, Mingyuan H, Jianping D, Zhanhong Z, Yong L (2005) Isolation of photosynthetic bacteria hp-1 with degradation of organic-phosphorus insecticides and studies on its biodegradation ability and capacity of increasing growth. Life Sci Res 3:247–253. https://doi.org/10.16605/j.cnki.1007-7847.2005.03.011
Doyle J (1991) DNA protocols for plants-CTAB total DNA isolation. Mol Tech Taxon. https://doi.org/10.1007/978-3-642-83962-7_18
Errampalli D, Leung K, Cassidy MB, Kostrzynska M, Blears M, Lee H, Trevors JT (1999) Applications of the green fluorescent protein as a molecular marker in environmental microorganisms. J Microbiol Methods 35(3):187–199. https://doi.org/10.1016/S0167-7012(99)00024-X
Flint HJ, Bisset J, Webb J (1989) Use of antibiotic resistance mutations to track strains of obligately anaerobic bacteria introduced into the rumen of sheep. J Appl Microbiol 67(2):177–183. https://doi.org/10.1111/j.1365-2672.1989.tb03393.x
Gotz M, Gomes NCM, Dratwinski A, Costa R, Berg G, Peixoto MHL, Smalla K (2006) Survival of gfp-tagged antagonistic bacteria in the rhizosphere of tomato plants and their effects on the indigenous bacterial community. FEMS Microbiol Ecol 56:207–218. https://doi.org/10.1111/j.1574-6941.2006.00093.x
Inouye S, Tsuji FI (1994) Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett 341(2–3):277–280. https://doi.org/10.1016/0014-5793(94)80472-9
Inui M, Nakata K, Roh JH, Zahn K, Yukawa H (1999) Molecular and functional characterization of the Rhodopseudomonas palustris No. 7 phosphoenolpyruvate carboxykinase gene. J Bacteriol 181(9):2689–2696
Koch B, Jensen LE, Nybroe OA (2001) Panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site. J Microbiol Methods 45(3):187–195. https://doi.org/10.1016/S0167-7012(01)00246-9
Lagendijk EL, Validov S, Lamers GEM, Weert SD, Bloemberg GV (2010) Genetic tools for tagging gram-negative bacteria with mCherry for visualization in vitro and in natural habitats, biofilm and pathogenicity studies. FEMS Microbiol Lett 305(1):81–90. https://doi.org/10.1111/j.1574-6968.2010.01916.x
Lewis PJ, Marston AL (1999) GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene 227(1):101. https://doi.org/10.1016/S0378-1119(98)00580-0
Li YQ, Kan ZR, Mu SM (2005) Study progress of photosynthetic bacteria. J Hebei Univ 25(5):554–560
Madhaiyan M, Poonguzhali S, Ryu JH, Sa TM (2006) Regulation of ethylene levels in canola (Brassica campestris) by 1-aminocyclopropane-1-carboxylate deaminase-containing Methylobacterium fujisawaense. Planta 224:268–278. https://doi.org/10.1007/s00425-005-0211-y
Nairn JD, Chanway CP (2002) Temporary loss of antibiotic resistance by marked bacteria in the rhizosphere of spruce seedlings. FEMS Microbiol Ecol 40(3):167–170. https://doi.org/10.1016/S0168-6496(02)00230-1
Niedenthal RK, Riles L, Johnston M, Hegemann JH (1996) Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast. Yeast 12(8):773–786. https://doi.org/10.1002/(sici)1097-0061(19960630)12:8%3c773:aid-yea972%3e3.0.co.2-l
Nunkaew T, Kantachote D, Kanzaki H, Nitoda T, Ritchie RJ (2014) Effects of 5-aminolevulinic acid (ALA)-containing supernatants from selected Rhodopseudomonas palustris strains on rice growth under NaCl stress, with mediating effects on chlorophyll, photosynthetic electron transport and antioxidative enzymes. Electron J Biotechnol 17(1):19–26. https://doi.org/10.1016/j.ejbt.2013.12.004
Omer ZS, Tombolini R, Gerhardson B (2004) Plant colonization by pink-pigmented facultative methylotrophic bacteria (PPFMs). FEMS Microbiol Ecol 47(3):319–326. https://doi.org/10.1016/S0168-6496(04)00003-0
Park JM, Yusin J, Kim TY, Lee SY (2010) Development of a gene knockout system for Ralstonia eutropha H16 based on the broad-host-range vector expressing a mobile group II intron. FEMS Microbiol Lett 309(2):193–200. https://doi.org/10.1111/j.1574-6968.2010.02041.x
Pelletier DA, Hurst GB, Foote LJ, Lankford PK, Mckeown CK, Lu TY, Schmoyer DD, Shah MB, Hervey WJ, McDonald WH, Hooker BS, Cannon WR, Daly DS, Gilmore JM, Wiley HS, Auberry DL, Wang YS, Larimer FW, Kennel SJ, Doktycz MJ, Morrell-Falvey JM, Owens ET, Buchanan MV (2008) A general system for studying protein-protein interactions in Gram-negative bacteria. J Proteome Res 7(8):3319–3328. https://doi.org/10.1021/pr8001832
Pleban S, Ingel F, Chet I (1995) Control of Rhizoctonia solani and Sclerotium rolfsii in the greenhouse using endophytic Bacillus spp. Eur J Plant Pathol 101(6):665–672. https://doi.org/10.1007/BF01874870
Poonguzhali S, Madhaiyan M, Yim WJ, Kim KA, Sa TM (2008) Colonization pattern of plant root and leaf surfaces visualized by use of green-fluorescent-marked strain of Methylobacterium suomiense, and its persistence in rhizosphere. Appl Microbiol Biotechnol 78(6):1033–1043. https://doi.org/10.1007/s00253-008-1398-1
Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of the Aequorea Victoria green-fluorescent protein. Gene 111(2):229–233. https://doi.org/10.1016/0378-1119(92)90691-H
Remus-Emsermann MNP, Schlechter RO (2018) Phyllosphere microbiology: at the interface between microbial individuals and the plant host. New Phytol 218(4):1327–1333. https://doi.org/10.1111/nph.15054
Remus-Emsermann MNP, de Oliveira S, Schreiber L, Leveau JHJ (2011) Quantification of lateral heterogeneity in carbohydrate permeability of isolated plant leaf cuticles. Front Microbiol 2:197. https://doi.org/10.3389/fmicb.2011.00197.Remusemsermann
Remus-Emsermann MNP, Lücker S, Müller DB, Potthoff E, Daims H, Vorholt JA (2014) Spatial distribution analyses of natural phyllosphere-colonizing bacteria on arabidopsis thaliana revealed by fluorescence in situ hybridization. Environ Microbiol 16(7):2329–2340. https://doi.org/10.1111/1462-2920.12482
Ruotian HE (2007) Research progresses of photosynthetic bacteria in plant culture. J Guangxi Agric Biol Sci 26(1):76–82
Sandhu A, Halverson LJ, Beattie GA (2007) Bacterial degradation of airborne phenol in the phyllosphere. Environ Microbiol 9:383–392. https://doi.org/10.1111/j.1462-2920.2006.01149.x
Shishido M, Breuil C, Chanway CP (2010) Endophytic colonization of spruce by plant growth-promoting rhizobacteria. FEMS Microbiol Ecol 29(2):191–196. https://doi.org/10.1111/j.1574-6941.1999.tb00610.x
Song HG (2007) Effects of application of Rhodopseudomonas sp. on seed germination and growth of tomato under axenic conditions. J Microbiol Biotechnol 17(11):1805–1810. https://doi.org/10.1016/j.mimet.2007.08.008
Su P, Tan XQ, Li CG, Zhang DY, Cheng JE, Zhang SB, Zhou XG, Yan QP, Peng J, Zhang Z, Liu Y, Lu XY (2017) Photosynthetic bacterium Rhodopseudomonas palustris GJ-22 induces systemic resistance against viruses. Microb Biotechnol 10(3):612–624. https://doi.org/10.1111/1751-7915.12704
Talbot NJ, Ebbole DJ, Hamer JE (1993) Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5:1575–1590. https://doi.org/10.2307/3869740
Tao S, Li N, Zaho G (2013) Livestock and poultry manure water treatment by two-stage fluidized beds formed with photosynthetic bacteria. Technol Water Treat 39(5):97–99
Teh BS, Apel J, Shao Y, Boland W (2016) Colonization of the intestinal tract of the polyphagous pest Spodoptera littoralis with the gfp-tagged indigenous gut bacterium Enterococcus mundtii. Front Microbiol. https://doi.org/10.3389/fmicb.2016.00928
Tombolini R, Unge A, Davey ME, Bruijnbcd FJ, Janssona JK (1997) Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescens bacteria. FEMS Microbiol Ecol 22(1):17–28. https://doi.org/10.1111/j.1574-6941.1997.tb00352.x
Torres AR, Araújo WL, Cursino L, Rossetto PB, Mondin M, Hungria M, Azevedo JL (2013) Colonization of Madagascar periwinkle (Catharanthus roseus) by endophytes encoding gfp marker. Arch Microbiol 195(7):483–489. https://doi.org/10.1007/s00203-013-0897-3
Van Loon LC (2007) Plant responses to plant growth-promoting rhizobacteria. Eur J Plant Pathol 119:243–254. https://doi.org/10.1007/s10658-007-9165-1
Wang YB, Li JR, Lin J (2008) Probiotics in aquaculture: challenges and outlook. Aquaculture 281:1–4. https://doi.org/10.1016/j.aquaculture.2008.06.002
Wang B, Li FF, Huang CJ, Yang XL, Qian YJ, Xie Y, Zhou XP (2014) V2 of tomato yellow leaf curl virus can suppress methylation-mediated transcriptional gene silencing in plants. J Gen Virol 95:225–230. https://doi.org/10.1099/vir.0.055798-0
Xiong MH, Hu ZG, Zhang Y, Cheng XS, Li CY (2013) Survival of GFP-tagged Rhodococcus sp. D310-1 in chlorimuron-ethyl-contaminated soil and its effects on the indigenous microbial community. J Hazard Mater 253(4):347–354. https://doi.org/10.1016/jhazmat.2013.02.54
We are thankful to the Hunan Engineering Laboratory for their support, especially to Dongwei Wang and Luyun Luo in data processing. In addition, the authors also thank Mr. Xinshun Ding for helping in language.
This research was supported by the National Key R&D Program of China (2017YFD0200400), the National Science Foundation of China (31701764), Hunan agricultural science and technology innovation fund (2017GC04), Hunan Natural Science Foundation (2017JJ3169), and the Hunan Provincial key research and Development Plan (2016NK2199). The grant agencies had no role in study design, data collection and analysis, the decision to publish, or the preparation of this manuscript.
Ethics approval and consent participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1.
Single cell expression of GFP tagged strain R. palustris GJ-22.
Additional file 2.
The sequences of gene of gfpmut3a and pckA.
Additional file 3.
The sequence of recombinant plasmid pBBR1-pckAPT-gfp.
Rights and permissions
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
About this article
Cite this article
Zhai, Z., Du, J., Chen, L. et al. A genetic tool for production of GFP-expressing Rhodopseudomonas palustris for visualization of bacterial colonization. AMB Expr 9, 141 (2019). https://doi.org/10.1186/s13568-019-0866-6
- Rhodopseudomonas palustris
- Green fluorescent protein