Filamentous phages belong to the genus Inovirus, members of which infect almost exclusively Gram-negative bacteria. The virions of these phages are slender filaments usually about 6 nm in diameter and 800-2000 nm long, comprising a helical capsid of several thousand copies of the major coat protein (pVIII) surrounding a single-stranded circular DNA genome about of 5-8 kb, encoding around 10 genes (Marvin, 1998). These phages usually use type IV pili as receptors to infect bacterial cells (Marvin, 1998).
Infection of bacteriophage to the host cells causes many change in morphology, physiology and genetics. Webbs et al. (2004) found that Pseudomonas aeruginosa, a human lung pathogen, to be form small colony variant (SCV) caused by Pf4 phage infection. Infection of Pf5 phage was reported to be increase the virulence of P. aeruginosa (). Moreover, virulence factor of pathogenic bacterias have been shown to be encoded by prophage (Brussow et al., 2004). CTX phage was reported to be increase the virulence of host bacteria, Vibrio cholera, by promoting CT toxin that affect to the development cholera disease (Davis and Waldor, 2003). Indeed, the define exotoxin from pathogenic bacteria, Shiga toxin by Escherichia coli (Johannes and Römer, 2010); botulism toxin by; diptheria toxin by , have been show to be expressed from phage-encoded gene.
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In Phytopathogenic bacteria, phages have been reporting to infects and affects the virulence of host bacteria. Filamentous phage Xf and Xf2 were reported to infect Xanthomonas campestris pv oryzae NP5850 and caused increasing virulence due to overproduction of extracellular polysaccharides (Kamiunten and Wakitomo, 1982). Moreover, Tseng et al. (1990) reported that filamentous phage Lf which infect X. campestris pv campestri increased virulence by promoting EPS production.
Recently, Yamada et al. (2007) have been succeed to isolate various bacteriophage that infect phytopathogen, Ralstonia solanacearum. Moreover, Kawasaki et al. (2008) characterized the genome of two kind of Ff-like phage (inoviridae),RSS1 and RSM1, were have host genome integration activity. Askora et al. (2009) also found a Ff-like phage, RSM3 and RSM4, that are enhance bacterial cell aggregation and reduce the bacterial host virulence. However, detailed information of cell aggregation and reducing virulence is unclear.
R. solanacearum, a widely distributed soil-borne pathogen belonging to the β subdivision of Proteobacteria, causes a lethal wilting disease of more than 200 plants species including economically important crops (Hayward, 2000). During infection, bacterial express virulence and pathogenicity factors resulting in typical wilting symptoms. Virulence and pathogenicity of R. solanacearum was known controlled by T2SS and T3SS (Poueymiro and Genin, 2009). As virulence factors, R. solanacearum produce consortium of plant cell wall-degrading enzymes (CWDEs), which are secreted via the type II secretion system (T2SS), such as β-1,4-endoglucanase (Egl), endopolygalacturonase (PehA), exopolygalacuturonases (PehB and PehC), β -1,4-cellobiohydrolase (CbhA) and a pectin methyl esterase (Pme) (Denny et al., 1990; González and Allen 2003; Huang and Allen 2000; Tans-Kersten et al., 1998). Moreover, secretion of protein (T3Es) via Type III Secretion System (T3SS) was known as important factor of bacteria pathogenicity. Loss of these secretions causes the loss ability to infect host-plant (Genin et al., 2005). During pathogen infection into plant host, plant will enhance and alter some physiological chance to challenge pathogen infection such as expression of pathogenesis-related gene that encode pathogenesis related protein (PR-protein). Hase et al. (2006) showed that Tomato pathogen infection, Pythium oligandrum, was shown high expression of PR-2b, PR-3b and PR-5b gene of plant which related to the expression of PR-protein. Moreover, Aime et al. (2008) compared infection tomato plant with virulence and avirulent strain of Fusaium oxysporum which showed high accumulation of 5 PR-protein in root and leaves of tomato inoculated with virulent strain compared with avirulent strain.
In this study, we showed an evidance of avirulent formation of R. solanacearum after infection by filamentous phage RSM3.This study was conducted both in bacterial cell and in the interaction of host-pathogen.
Colony Morphology, Culture Growth and Aggregation Assay
To observe the culture color, a single colony of Twenty four house cultures in CPG Agar of Ralstonia solanacearum was picked-up and transfer to CPG broth and was incubated in 28OC with shaking 250-300 rpm until 3 days. Changing of culture color was recorded every day. Culture characteristic on CPG agar was determined by streak plate of OD600 1.0 of 24 hours bacterial culture. To obtain a single colony size, a twenty four hours culture was recovered by centrifugation and was washed two times with ddH2O and was adjusted OD600 of 0.1 and was streaked onto CPG and MM agar. Observation was done after 48 hour of incubation in 28OC.
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Twenty four hours cultures in CPG of R. solanacearum was adjusted to OD600 of 0.01 as initial OD with CPG Broth. The culture was incubated in 28OC with shaking 250-300 rpm and was measured the OD600 every 1 hour grown for 1 day at 28OC. Curve of bacterial growth was calculated using absorbance on OD600 and time of incubation. To observe the change of characteristic of liquid culture, bacteria was cultured in liquid CPG for 3 days and observed for color change of liquid CPG medium.
Aggregation assay was performed from Bacterial cell from Twenty four hours cultures in CPG bya harvested cells by centrifugation at 15,000 x rpm for 2 min at 4°C, washed three times with ddH2O and adjusted the OD660 of 1.0 with 1 x PBS pH 7.0. Suspension was incubated without agitation in 28OC. absorbance at 660 nm of upper suspension (100 µL) was measured after 0, 0.5, 1, 2, 4, and 6 h of incubation. Percentage of aggregation curves were then constructed following the formula (Eboigbodin et al, 2005).
% Aggregation= OD0- ODtOD0 x 100
where OD0 is the optical density at 660 nm of bacteria immediately after resuspended with 1 x PBS and ODt is the optical density after particular time.
Cultures of R. solanacearum were grown for 1 day at 28OC in CPG broth. Bacterial cells were obtain by centrifugation at 15.000 rpm, 4OC, for 2 minutes and were washed with ddH2O two times. Pellet was resuspended with ddH2O and was adjusted the OD600 to 1.0. Fifty microliter of suspension was dropped into test media for each assay (MM for twitiching motility, SWM for swimming motility and SRM for swarming motility). Motility was observed by measuring the diameter of dropped-culture up to 6 days.
To test the movement in tomato stem, GFP-expressing strain was used in the experiment. Both strains, MAFF106603 contain pRSS12 and its infected cell by ΦRSM3 was grown in CPG media for 24 hours. Bacterial cells were obtain by centrifugation at 15.000 rpm, 4OC, for 2 minutes and were washed with ddH2O two times. Pellet was resuspended with ddH2O and was adjusted the OD600 to 1.0. One microliter of suspension was injected into tomato stem between cotyledon and the first leaf and incubated at 28OC. After 1 week incubation, plant stem was cut 20 µm in thickness with microtome and then observed using a Leica MZ16 microscope with GFP3 filter.
Quantification Endoglucanase Activity and Extracellular Polysaccharides
Total endoglucanase activity was quantified by measuring the reduction sugar (Nelson, 1944) released during incubation at 50OC for 4 hours with 4 mg/ml of carboxyl metylcellulose in 120 mM phosphate buffer (pH 7.0) In soluble material was removed by low speed centrifugation before absorbency readings were taken on 540 nm. One unit of enzyme activity was defined as releasing 1 nmole/minute of glucose.
Cultures of R. solanacearum were grown for 3 days at 28OC in EG broth (Denny et al., 1990). To recover EPS, culture supernatants were adjusted to 0.1 M NaCl and 4 volume of acetone were added. After incubating overnight at 4OC, precipitated material was recovered by centrifugation (15.000 rpm, 10 minutes, 4OC), dissolved in 200 µl of ddH2O, heated at 65OC for 10 minutes and centrifuged for 5 minutes to remove in soluble material. The concentration of hexosamine in culture supernatants was estimated with a modified Elson and Morgan reaction. Appropriately diluted samples (0.45 ml) were mixed with 0.15 ml of concentrated HCl, hydrolyzed at boiled water (110OC) for 30 minutes in sealed tube and the colorimetric assay performed. The results were read at OD530, the background due to residual media components substraced, and the hexosamine concentration determined from N-acetyl D-glucosamine standard curve. N-acetyl D-glucosamine standards were subjected to the entire analysis procedure beginning with hydrolysis step.
Bacterial Surface appendages
Cells of R. solanacearum strains were streaked heavily on minimal medium plates (Clough et al., 1994) and incubated for about 22-24 hours. The colonies were washed off in a small volume of 10 mM Tris-HCL buffer pH 8, and the cell suspension was forced five times through a 25-gauge hypodermic needle. Bacterial cells were removed by centrifugation with a R12A2 rotor in a Hitachi himac CR21E centrifuge at 8,000- rpm for 20 min at 4°C. The bacterial surface appendages were collected by ultracentrifugation with a R12A2 rotor in a Hitachi himac CR21E centrifuge at 136,000-g for 60min. Precipitated exostructure were subjected to Tris-Tricine PAGE according to Schagger and von Jagow (1987).
Total RNA preparation
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All strains, R. solanacearum MAFF106603 and its infected cell by ΦRSM3, were grown in Minimal Medium (MM) at 28OC for 24 hours. The new sub culture were made by adjusting the OD600 to 0.02-0.03 in new MM and continued growing to reach OD600 of 0.1.
Total RNA was prepared from 3 ml of the exponential phase culture with RNeasy Mini columns (Qiagen), and was eluted in 60 µl of DEPC-treated water. The total RNA was treated with Recombinant DNase I (Takara) to remove any genomic DNA contamination by incubation with 10 U of RNase-free DNase I for 30 min at 37°C. The DNaseI was inactivated by phenol/chloroform extraction method. To confirm the presence of DNA contaminant, a 30 PCR cycles was performed using a gene specific primer (Table 1.) with DNA genome of MAFF106603 as a positive control.
The cDNA of target genes was synthezed by using M-MLV Reverse Transcriptase RNase H- (ReverTra Ace, Toyobo, Osaka). Specific primers were designed using the Primer3 (v.0.4.0) software (http://frodo.wi.mit.edu/primer3/#PRIMER_MAX_TEMPLATE_MISPRIMING) and were checked for gene specificity using DDBJ/Blast (v.2.2.18). Breafly, 20 μl of Reaction Mixture (5 μl of 0.1-1 μg total RNA, 1 μl of 5 pmoles of gene spesific primer, 4 μl of 5X ReverTra Ace Buffer, 2 μl of 10 mM dNTPs, 1 μl of 100 units ReverTra Ace and 7 μl of DEPC-treated water) was incubated for 60 minutes at 42OC and was inactivated by incubation at 99OC for 5 minutes. Negative control reactions to eliminate the possibility that residual DNA was amplified were performed in the same way, except that the RT was omitted from the reaction mixtures.
Real Time-Quantitative Polymerase Chain Reaction (Real Time-qPCR)
Real-time PCR was performed using a Line Gene Fluorescence quantitative detection system (BioFlux, Tokyo) with cDNAs prepared from R. solanacearum MAFF106603 strains. A PCR mixture containing SYBR-green (SYBR premix ExTaq, Takara Shuzo, Kyoto) was used. PCR reactions in a final volume of 10 μl reaction mixture containing 5 μl PCR mixture, 1 μl diluted cDNA and 0.5 μM each primer (Table 1.) were carried out under the following conditions: 3 min at 95°C and 45 cycles at 95°C for 10 s, 62°C for 10 s, and 72°C for 15 s. At the end of the program, the specificity of the primer set was confirmed by melting curve analysis (65-95°C with a heating rate of 0.5°C/min). The copy numbers of spesific target gene and 16s rRNA were estimated by comparing the results of real-time PCR with several dilutions (102, 103, 104, 105 copies/μl) of each gene. The mRNA level of 16s rRNA was used to normalize the expression ratio of each gene.
Table 1. Primers used in real-time qRT-PCR study
Forward primer 5′-3′
Reverse primer 5′-3′
CAG CGC GAC CTA CTA CAA GA
TCA TCA GCC CGA AGA TGA C
TTC TCG ATG ATG TAG CGA TAG G
GCT GGA ATT TCG ACT TCC TCT A
CTA CCA GAT CGT CGT CAA TGA A
GTC GAG GTA GTG CTT GAT CTT G
GTT GTT CGG ATT GCT GTA CG
AGT CAA ACG ATT GCC TGA ACT A
CTA GAG TGT GTC AGA GGG AGG TAG A
ATG TCA AGG GTA GGT AAG GTT TTT C
Note. Bolded characters of oligo primer sequence refers to the Primer used in cDNA synthesis.
Table 2. The tested genes and their putative functions
Endoglucanase, enzyme which involved in degradation of plant cell wall glucans. (Roberts et al. 1988)
Regulator the expression of T3SS biosynthesis genes, and probably >60 effector substrates, and T3SS-dependent export pathway (Occhialini et al., 2005).
Requiring for production of an extracellular factor (EF), identified as the fatty acid derivative 3-hydroxypalmitic acid methyl ester (3-OH PAME), which function as autoinducer (quorum sensing signal) in R. solanacearum (Flavier et al., 1997a, Flavier et al., 1997b)
Exopolygalacturonase (Gonzalez and Allen., 2003).
Encode 16S ribosomal RNA.
Virulence assay and HR test
Tomato plants were cultivated in 5-cm pots filled with a soil-peat mixture. Plants (4 leaves) were selected for inoculation. Twenty four hours bacterial culture was centrifuged at 15.000 rpm, 4OC, for 2 minutes and were washed with ddH2O two times. Pellet was resuspended with d ddH2O and was adjusted the OD600 to 1.0. One microliter of suspension was injected into 6 weeks old tomato stem between cotyledon and the first leaf and incubated at 28OC. As a control, distilled water was injected in the same manner. Disease index was observed following Poueymiro et al. (2009) up to 1 week after inoculation with scale ranging from 0 to 4, according to the percentage of wilted plants (0= no wilt; 1 = 1 to 25%; 2= 26-50%; 3=51-75%; 4=>75%).
HR-eliciting ability was tested by infiltrating a serial dilution of R. solanacearum strain solution into the intercellular space of tobacco leaves (Nicotiana tabacum) (Kim et al., 1997). Plants were incubated in 25OC and hypersensitive response was observed 24-48 hours after infiltration.