Hemin transported protein of Xanthomonas axonopodis pv. glycines functions on leaf colonization and virulence on soybean

Xanthomonas axonopodis pv. glycines (Xag) causes bacterial pustule disease on soybean. This bacterium is present worldwide around hot and humid growing regions such as Southeast Asia. To understand if the gene coding for hemin transport protein (hem) is involved in virulence of the pathogen in soybean, we generated a hem mutant in Xag by overlapping PCR mutagenesis. Disruption of hem significantly reduced the population size and the disease incidence when sprayed on soybean but not when injected directly to soybean. The hem mutant caused the hypersensitive response induction on tobacco as an Xag wildtype. Interestingly, the hem expression was also reduced when the Xag wildtype grow in planta. The hemin transporter protein involved in the production of extracellular polysaccharide, biofilm formation, motility and attachment but not for extracellular enzymes. This confirmed that epiphytic fitness of Xag strongly required hem functions. These results suggest that hem gene is essential for virulence of Xag on soybean during the infection process.


INTRODUCTION
Bacterial diseases of soybean appear worldwide and cause production losses and decreases yield by reducing quality and quantity.The most common bacterial disease of soybean is bacterial pustule, caused by Xanthomonas axonopodis pv.glycines, is one of the most serious diseases of soybean in several part of soybean production areas including Thailand.Bacterial pustule lesions are small pale green spots with raised centers on either or both leaf surfaces.The bacterial pustule lesions may enlarge and coalesce, leading to premature defoliation (Narvel et al., 2001).Severe disease causes yield losses up to 40% (Prathuangwong and Amnuaykit, 1989).X. axonopodis pv.glycines infects the soybean plant through stomata and wounds.After invasion into the plant, bacteria multiply within intercellular spaces of the spongy mesophyll for pustule induction on susceptible soybeans (Jones and Fett, 1985).
Nutritional conditions are reported to be an important virulence factors for the disease induction of plant pathogenic bacteria.For example, the translations of pathogenicity island (hrp gene cluster) of Xanthomonas are induced by sucrose and sulfur-containing amino *Corresponding author.E-mail: agrsdp@ku.ac.th.
acids (Schulte and Bonas, 1992).While iron is an essential element for pathogenic bacteria due to its participation in the tricarboxylic acid cycle, electron transport, amino acid and pyrimidine biosynthesis, DNA synthesis, and other critical functions (Lemanceau et al., 2009).Moreover, iron is considered to play a critical role in plant-bacterial interactions.During bacterial infection, there is aggressive competition between the plant and the bacteria iron can play a critical role in such competitive relationships (Lemanceau et al., 2009).Segond and collaborators found that metal transporter AtNRAMP3 in Arabidopsis is upregulated in leaves challenged with the Pseudomonas syringae and Erwinia chrysanthemi (Segond et al., 2009).Similarly, A. thaliana synthesizes the ferritin AtFER1, an iron storage protein, is required for Arabidopsis resistance to E. chrysanthemi infection (Boughammoura et al., 2007).
Iron is one of the factors that limits bacterial growth in planta because concentrations of iron are necessary to support bacterial growth and multiplication (Expert et al., 1996).Siderophore-mediated transport of iron is one of the mechanisms used by bacteria to uptake iron from their environment (Braun et al., 1998;Lee, 1995;Mietzner and Morse, 1994).Thus, the production of a siderophore by bacterial pathogens could significantly deplete the iron reserves of the host plant and weaken host-defence reactions (Lemanceau et al., 2009).Several studies have shown the importance role of iron in virulence of plant pathogenic bacteria.For instance, siderophore-deficient mutants of Erwinia amylovora, E. chrysanthemi strain 3937, Erwinia carotovora subsp.carotovora, Ralstonia solanacearum and Agrobacterium tumefaciens are virulence deficient on its host plants (Dellagi et al., 1998;Franza et al., 2005;Bhatt and Denny, 2004;Bull et al., 1996;Rondon et al., 2004).However, P. syringae pv.syringae B301D and P. syringae pv.tomato DC3000 do not show any growth defect or alter virulence on host plant (Jones et al., 2007;Jones and Wildermuth;2011).In addition to the uptake of iron, several Xanthomonas take up iron via tonB system.In Xanthomonas campestris pv.campestris, mutation of tonB, exbB and exbD1 genes which are involved in iron uptake system have been reported to be impaired for ferric ion uptake and exhibited reduced virulence in cabbage (Wiggerich and Puhler, 2000).The fur mutant of Xanthomonas oryzae pv.oryzae is virulence deficient and hypersensitive to oxidative stress (Subramoni and Sonti, 2005).Different pathways of iron uptake from direct Fe 2+ transport and host iron binding proteins or heme also may be employed by pathogenic bacteria (Ratledge and Dover, 2000;Velayudhan et al., 2000).
Hemin is one of heme oxidized form that consists of an iron ion and found in extracellular environments (Lee, 1995).Hemin iron transport and utilization systems have been identified in numerous bacterial species, where it was shown that an outer membrane receptor and a periplasmic binding protein-dependent ABC-type transporter are required for hemin uptake (Stojiljkovic andHantke, 1992, 1994).It is the cofactor in reactions involved in various cellular functions including oxygen transport and electron transfer (Lee, 1995).
For phytopathogenic bacteria, Xylella fastidiosa 9a5c contains 67 genes encoding proteins involved iron metabolism and has been reported to contain five membrane receptors, including siderophore, ferrichromeiron and hemin receptors, all of which are thought to be associated with iron transport, utilization and virulence (Simpson et al., 2000).Whereas, the extensive genetic and genomic resources are available for X. axonopodis pv.glycines, and it has been the subject of highly productive research centered on the mechanisms of plant host susceptibility/resistance and pathogen virulence and avirulence determinants (Athinuwat et al., 2009;Chatnaparat et al., 2012;Kasem et al., 2007;Kaewnum et al., 2005;2006;Thowthampitak et al., 2008).Recently, the draft genome of X. axonopodis pv.glycines 12-2 has been sequenced and found that this strain contains genes encoding hemin uptake locus.Although, the effects of iron uptake system in cell growth and virulence production of several bacteria plant pathogens have been documented, hemin transport protein (hem) of the bacteria that involved in the infection process of plant are not established.Thus, in this study, a hem mutant was constructed using overlapping extension mutagenesis.The roles of hem in contribute to full virulence of Xag on soybean were investigated.

Bacterial strains, plasmids and recombinant techniques
Bacterial strains and plasmids used in this study are described in Table 1.X. axonopodis pv.glycines wildtype strain 12-2 was cultured at 28°C in nutrient glucose agar (NGA) (Sambrook et al., 1989).Mutants were cultured on NGA containing 50 g/ml kanamycin and 50 g/ml chephalexin.The complemented hem was cultured on NGA containing 50 g/ml kanamycin, 50 g/ml cephalexin, and 40 g/ml gentamycin.All DNA manipulations including DNA isolation, plasmid extraction, restriction digestion, ligation, and gel electrophoresis were performed as described previously (Sambrook et al., 1989).

Knockout of hem genes in X. axonopodis pv. glycines 12-2
A disruption of gene coding for hemin transport protein (hem) was accomplished using overlap extension mutagenesis (Figure 1).The upstream and downstream regions of hem gene in X. axonopodis pv.glycines 12-2 were amplified using HemKO-1-F and HemKO-1-R primers that unique to upstream region and HemKO-2-F and HemKO-2-R primers that unique to downstream region of hem gene respectively (Table 2), with one having an extension complementary to the kanamycin resistance cassette from pKD13 to generate two amplicons with ends overlapping those of the resistance cassette (Datsenko and Wanner, 2000).Overlap This study extension PCR was used to link the two PCR amplicons and the resistance cassette; this larger fragment was then cloned into the destination vector pTok2 using the quick ligation protocol (New England Biolabs Inc.) and introduced into E. coli S17-1, the mobilizing strain by transformation, then transferred to X. axonopodis pv.glycines via conjugation, selecting for transconjugants on NGA containing 50 g/ml kanamycin and 50 g/ml chephalexin as a sensitive antibiotic for E. coli strain.Gene disruption was confirmed using PCR, with primers specific (Hem-C-F and Hem-C-R) to the sequences flanking of hem gene (Table 2).

Complementation of hem mutants
To complement the hem mutant, 800 bp of hem containing the native promoter was amplified using primers hem com-F and hem com-R (Table 2).The amplicon was digested with HindIII and ligated into the multiple cloning site of vector pBBR1MCS-5 to yield pBBR::hem, which was then introduced into hem mutant by electroporation.The complemented hem mutant was cultured on NGA containing 50 g/ml kanamycin, 50 g/ml cephalexin, and 40 g/ml gentamycin and also confirmed by PCR using primers hem com-F and hem com-R.

Real-time quantitative reverse transcription PCR analysis
The expression of hem was determined by real-time qRT-PCR of cDNA isolated from both an Xag wildtype and a hem mutant grown in nitrogen yeast glycerol broth (NYGB) for 24 h as well as in X. axonopodis pv.glycines cells recovered from infected soybean plants.For in planta experiment, Xag cells in soybean were isolated from the leaves according to the method described by Yu et al. (2013).Briefly, the bacterial cells at 1 × 10 8 cfu/mL were introduced by vacuum infiltration into soybean leaves.Infiltrated plants were incubated for 4 days.A total of 150 to 200 leaves were collected cut into squares, and submerged in an acidic phenol RNA-stabilizing solution.The solution was filtered and centrifuged to harvest the bacterial pellets.Total RNA preparation for using in the real-time qRT-PCR was isolated with TRIzol (Invitrogen Life Technologies) from cells grown in NYGB and in planta using the method of Santiago-Vazquez and associates (2006).cDNA was generated from 1 μg of RNA using SuperScript II (Invitrogen Life Technologies, Carlsbad, CA, U.S.A.) and random hexamers.Real-time qRT-PCR was performed on 1 μg of the cDNA using LightCycler FastStart DNA Master PLUS SYBR Green I (Roche, Indianapolis, IN, U.S.A.) on a Roche Lightcycler II (Roche) following the manufacturer's specifications.The specific primer pairs in this experiment are list in the Table 2.An external standard curve was generated using purified ihfA (integration host factor A) DNA (Champoiseau et al., 2006).Melting curve analysis was used to verify amplification of a single product.The concentration of amplification products from negative controls (RNA samples to which no superscript was added) was undetectable in all cases, indicating a lack of interference from contaminating DNA.

High iron concentration sensitivity assay
To test for high iron concentration sensitivity, an Xag wildtype, a hem mutant and a complemented hem mutant were grown in NYGB at 28°C with shaking at 200 rpm to an optical density (OD) at 600 nm of 1.0 (OD600 = 1.0).Cultures were transferred to NYGB supplemented with FeCl3 to a series of final concentrations at 0, 4, 5, and 6 mM, respectively and were incubated at 28°C with shaking at 200 rpm.After incubation for 24 h, the cell density was measured spectrometrically using a spectrophotometer and absorbance at OD600 was determined (Yang et al., 2007).The experiments were repeated three times with at least three replicates in each experiment.

The extracellular polysaccharide (EPS) production
EPS production of an Xag wildtype, a hem mutant and a complemented hem mutant was measured with some modification as described by Tang et al. (1991).Cultures were grown in NYGB containing 4% glucose at 28°C with shaking at 200 rpm for 5 days.EPS was precipitated from the culture supernatant with ethanol.
Then EPS was dried at 80°C to constant weight and the difference between the two weights was used to estimate the production of EPS per millilitre culture.The experiments were repeated three times with at least three replicates in each experiment.

Biofilm formation
Cells of X. axonopodis pv.glycines strains taken from cultures grown on nitrogen yeast glycerol agar (NYGA) for 24 h were suspended in NYGB and cell suspension of each strain was added
to glass tubes, and grown at 28°C for three days.The presence of a biofilm was visualized as a white ring on the tube side wall, usually at the air-medium interface and quantified by crystal violet staining as previously described (Davey and O'Toole, 2000).Dye abundance was measured by absorption at 570 nm using a spectrophotometer.Readings from five replicates were averaged.The experiments were repeated three times with similar results.

Bacterial attachment
Bacterial adhesion of X. axonopodis pv.glycines strains to soybean leaves was assessed by immersing six individual leaves into 500 ml of a suspension of a given bacterial strain (10 7 cells/ml) at 28°C.After 5 min, 3 and 7 h, the leaves were removed and rinsed gently with distilled water for 30 s.To enumerate the attached bacteria a single 2 cm diameter disc was cut from a portion of each leaf in an area lacking major veins using a cork borer, the discs homogenized using a mortar and pestle, and cells enumerated by dilution plating on NYGA as in other studies (Chatnaparat et al., 2012).The experiments were repeated three times.

Motility analysis
Fresh colonies of an Xag wildtype, a hem mutant, and a complemented hem mutant from NYGA plates were stabbed into swarm and swimming plates composed of 0.03% (wt/vol) Bacto Peptone, 0.03% yeast extract, and 0.4% agar for swarm plate and 0.25% agar for swimming plate respectively.The inoculated cells were cultured for four days or longer at 28°C and examined for bacteria motile away from the inoculated site (Sockett and Armitage, 1991).The experiments were repeated three times and each experiment was measured in triplicate.

Extracellular enzymes assay
Relative levels of extracellular production including carboxymethylcellulase, α -amylase and protease were assessed by radial diffusion assays (Thowthampitak et al., 2008).The experiments were repeated three times and each experiment was measured in triplicate.
For carboxymethylcellulase production, inoculated plates containing an assay medium (0.1% carboxymethyl cellulose, 25 mM sodium phosphate, pH 7.0, and 0.8% agarose) were incubated at room temperature overnight, stained with 0.1% Congo red for 20 min, and washed twice with 1 M NaCl.Carboxymethyl cellulase (CMCase) activity was visualized as white halos surrounding the wells.
For protease production, inoculated plates containing NYGA supplemented with 0.5% skimmed milk were incubated at room temperature for 48 h.Extracellular protease production was detected visually as clear halos surrounding the wells.

Hypersensitive response (HR) and virulence assay
The X. axonopodis pv.glycines strains were grown in NYGB at 28°C with shaking at 200 rpm.Cells were pelleted at early log phase by centrifugation at 6,000 rpm for 2 min.Cell pellets were suspended in sterile water for HR tests on tobacco.HR was assayed as described previously (Kaewnum et al., 2005).Briefly, tobacco plants were inoculated with bacterial suspensions (10 9 cells/ml) by injection of leaf with a syringe.Sterile demineralized water was used as a negative control.Infiltrated zones were observed for development of typical HR (tissue collapse and necrosis) for 24 to 48 h post-infiltration.The experiments were repeated three times with similar results.
The virulence of X. axonopodis pv.glycines strains was assessed on susceptible soybean cv.Spencer following topical spray application (Kaewnum et al., 2005).Briefly, cell suspensions of a given strain (OD600 = 0.2; ca. 10 8 cells/ml) in 1 mM KPO4 buffer were sprayed onto leaves of plants (ca.6 weeks old) maintained in a greenhouse (average temperature ca.28°C).For the first 24 h after inoculation, plants were held in an enclosed plastic bag to maintain high humidity and moisture on leaves before being returned to the greenhouse bench.Three trifoliate leaves, collected each from the top, middle and basal portion of three plants from each of five replicate pots, were evaluated for each strain.
Cotyledon assay was done as described by Hwang et al. (1992).The 7-days old soybean seedling grown in a greenhouse was surface sterilized with 0.5% sodium hypochloride for 3 min and washed with sterile distilled water for 5 min.The cotyledon was punctured with sterile pins.10 l of each suspension of bacterial cells (10 8 cells/ml) of the wildtype, hem mutant, and complemented hem mutant were dropped on the wound site.Inoculated cotyledons were kept in high moisture conditions with 16h photo period at room temperature.The cotyledons were observed by chlorotic and necrotic symptoms around the inoculation site within 48 h after inoculation.At least five soybean cotyledons were used for each strain.The experiments were repeated three times with similar results.

Bacterial population on soybean leaf surface
For determination of epiphytic fitness, bacterial populations were isolated from the soybean leaves according to the modify method described by Morris et al. (1998).Inoculation of X. axonopodis pv.glycines strains on 6 weeks old soybean were designed to analyze the epiphytic fitness of the hem mutant in comparison to that of the Xag wildtype.Bacterial cultures were prepared to a final concentration of 4.5 × 10 5 cells per ml and then 50 ml of bacterial suspension was used to spray on soybean leaves as described above in virulence assay.Four pots containing 5 soybean plants in each pot were used for each strain.The plants were transferred to the greenhouse bench.Four leaves of inoculated soybeans were taken randomly from each treatment.Estimation of each bacterial numbers of the Xag wildtype, hem mutants, and complemented hem mutants were collected at 1, 3, 7 and 14 days after inoculation.The experiments were repeated two times.

Genetic characterization of the hem locus
Analysis of the DNA sequence of X. axonopodis pv.glycines 12-2 draft genome (GenBank accession number AJJO01000000) revealed the presence of the genes predicted as hemin uptake (hem) locus including hemin uptake protein, hemin uptake system outer membrane receptor, and hemin transport protein (hem), respectively.hem size is 800 bp encodes a protein of 211 amino acids.Hem shared the highest level of identity (99%) to the hypothetical protein of Xanthomonas axonopodis pv.citri 306 and Xanthomonas citri pv.mangiferaeindicae LMG 941, while it exhibited 80 and 70% identity to a hypothetical protein in X. campestris pv.vesicatoria 85-10 and X. campestris pv.campestris, respectively.However, protein predicted as hemin transport protein in X. axonopodis pv.glycines 12-2 shared similarity at 63 and 37% with hemin transport protein in Stenotrophomonas maltophilia D457 and Sinorhizobium fredii HH103 respectively.

High iron concentration sensitivity
The X. axonopodis pv.glycines wildtype and the hem mutant showed the same growth yield in NYGB without supplementation of FeCl 3 .When NYGB supplemented with FeCl 3 to final concentrations of 0, 4, 5, and 6 mM, respectively, significant differences in growth were observed between the Xag wildtype and the hem mutant.The Xag wildtype could grow well in NYGB supplemented with concentrations up to 5 mM, whereas the hem mutant decreased the growth under all conditions (Figure 2).The growth capacity of the hem mutant could be completely restored in the complemented hem mutant.

Pathogenicity and virulence assay
Leaf pathogenesis assay was conducted to determine the probable involvement of hem functions in bacterial virulence.The X. axonopodis pv.glycines wildtype strain, the hem mutants, and the complemented hem mutant were inoculated through injection to soybean cotyledons and through spray on soybean leaves.All strains developed normal disease symptoms as in its wildtype when inoculated through injection into the soybean cotylendons (Figure 3B).Interestingly, the virulence of the hem mutant appeared to be attenuated when the cells were applied on soybean leaves by spray inoculation (Table 3, Figure 3A).Moreover, the hypersensitive response activities of the hem mutant were similar to that of the Xag wildtype after infiltration into tobacco.This results suggest that hem gene is essential for virulence of X. axonopodis pv.glycines on soybean before penetrate into soybean plant.Thus, perhaps hem gene is important for epiphytic fitness of this  this pathogen.

hem gene expression
Real-time qRT-PCR was performed to determine transcript levels of hem gene in the X. axonopodis pv.glycines wildtype and the hem mutant in overnight culture in NYGB and also wildtype in soybean leaves after four days inoculation.Mutation in hem resulted in no expression of hem transcription in the culture medium confirming that the hem mutant strain completely lost hem gene.Furthermore, the level of hem transcript in the Xag wildtype cells recovered from infected soybean plants was 4.34 fold lower than that in the wildtype cells grown in culture (Figure 4).In addition, the expression of hem gene was not observed from cDNA of non-infected soybean.However, disruption of the hem gene did not affect the expression of the hrpF and hrpD genes, which are encoded in the hrp cluster when comparison with wildtype cells grown in Hrp inducing medium (data not shown).This results suggest that hem gene was down- regulated when bacteria grow in soybean plant and did not affect typeIII secretion system of this pathogen in vitro.Therefore, the hem mutants show a virtually indistinguishable disease symptom when compared to that of the wildtype strain when the cells were directly injected into soybean (by pass epiphytic fitness phase).

Bacterial population on soybean
The above data suggest that hem gene contributed virulence on soybean leave when spray inoculation but not when injected into soybean.Therefore, we assessed the ability of the hem mutants to grow on soybean leaf surfaces.As we expected, minimal cells number were observed in soybean leaves sprayed with the hem mutants (Figure 5).Taken together, these data indicate that hem gene is required for the leaf colonization of Xag on soybean.

Extracellular polysaccharide (EPS) production
The EPS production in three-day liquid cultures showed that hem mutants produced on average 0.62 mg per milliliter of the culture, compared with 1.0 and 1.1 mg per milliliter of the culture of wildtype and complemented hem mutants, respectively (Figure 6).EPS is an important virulence factor in X. axonopodis pv.glycines and in many pathogens (Braun, 1990;Thowthampitak et al., 2008).Our results showed the hem mutants decreased the production of EPS.Thus, we assumed that the reduction in the production of EPS may contribute to the deficiency in virulence of the mutant.

Biofilm formation
Biofilm formation is structure for protect the bacterial cell from stress environmental condition.Plant pathogenic bacteria within biofilms are generally better resistant to environmental stress and host defense response (Crossman and Dow, 2004).The biofilm formation of the hem mutants in NYGB as measured by crystal violet staining after 3 days of incubation was significantly lower than that of the wildtype and complemented hem mutants (Figure 7).Therefore, the hem mutants may not resistant to environmental stress such as dry conditions or UV and host defense response during pathogenesis.

Bacterial attachment
Since we found that hem gene effected to biofilm formation on abiotic surface.Therefore, the attachment of hem mutants to the soybean leaves surface was studied by quantifying those cells remaining on leaves after they were dipped into bacterial cell suspensions.The number of hem mutants cells was attachted soybean leaves surface significantly lower than the wildtype and complemented hem mutants (Figure 8).

Motility analysis
The hem mutant cells were swarming motile on semi-solid medium with 0.4% agar same as the wildtype and complemented hem mutant.However, we found that the swimming ability of the hem mutants on 0.25% agar swimming plate was significantly reduced when compared with the wildtype and complemented hem mutants (Figure 9).This result suggests that the hem mutants have effect to swimming motility but not for swarming movement.

Extracellular enzyme production
The production of extracellular proteases, amylases, and cellulase have been shown to be virulence factors in X. axonopodis pv.glycines 12-2 (Thowthampitak et al., 2008).To explore whether the hem mutant might also effects such virulence factors, these traits were compared among the wildtype, hem mutant, and complemented hem mutants in diffusion plate assay.We found that the expression of these extracellular enzyme productions did not differ among them (data not shown).

DISCUSSION
The acquisition of iron is thus one of the most important adaptive responses for bacterial pathogens.The ability of pathogenic bacteria to acquire iron from free heme and host hemoproteins has been studied by many laboratories for clinical pathogens (Braun et al., 1998;Lee and Levesque, 1997) but has not been reported in bacterial plant pathogens.We also found the sequence of hemin uptake locus system in draft genome of a bacterial pustule pathogen X. axonopodis pv.glycines 12-2 (GenBank accession number AJJO01000000).Sequence analysis of the hemin uptake locus revealed three genes including hemin uptake protein (Xag857), hemin uptake system outer membrane receptor (Xag858), and hemin transport protein (hem) required for use of hemin and hemoproteins as iron sources.In this study, we have analyzed the function of hem gene coding for hemin transport protein of X. axonopodis pv.glycines 12-2 that effect to virulence on soybean in the epiphytic phase of infection involving the extracellular polysaccharide (EPS) production, biofilm formation, attachtment, and motility.These might be suggesting that this hem system might be important for bacterial-plant interaction.
The colonization as epiphytic of X. axonopodis pv.glycines before infects through stomata or wounds on soybean leaves is very important process for cause pustule disease on soybean.In this report, the hem mutant of X. axonopodis pv.glycines was virulence deficient and decrease in the population size when sprayed on soybean plants but not when injected directly to soybean leaves.These results suggest that the hemin transport protein is essential in epiphytic phase, but not required for endophytic phase.Therefore, the reduction in virulence of the hem mutant is possible that hem might affect the expression of genes involving in the epiphytic fitness.Previous reports have indicated that hem is a multifunctional regulatory protein which controls the expression of trypsin-like protease, hemagglutinating, and hemolysin activities, as well as the production of extracellular vesicles in Porphyromonas gingivalis (Carman et al., 1990).Disruption of the hemin transport protein would result in the effected expression of these factors in X. axonopodis pv.glycines.This hypothesis is consistent with the regulation of hemin-responsive genes in bacteria by a negative regulator such as the welldescribed E. coli ferric uptake regulator (Fur).Fur acts as a classical negative regulator and uses Fe 2+ as a corepressor to bind the promoter region of iron-regulated genes (Bagg and Neilands, 1987).Regulation of ironregulated genes by a Fur-like system has been found in X. campestris pv.campestris and X. oryzae pv.oryzae plants (Jittawuttipoka et al., 2010;Subramoni and Sonti, 2005).The mutations in ferric uptake regulator were also resulted in the reduction in virulence of X. oryzae pv.oryzae and X. campestris pv.campestris on their host plants (Jittawuttipoka et al., 2010;Subramoni and Sonti, 2005).
In this study, the transcription level of hem was reduced when bacteria grow in soybean plant.After invasion into the leaf through stomata, bacteria multiply within the substomatal chambers and intercellular spaces of the spongy mesophyll (Jones and Fett, 1985).In the plant cells, iron concentration was higher than in the surface of the plant.Many iron and heme transport systems are repressed by iron and Fur under iron-rich conditions (Lee, 1995).A component of an iron-scavenging system (PSPTO2134) of P. syringae pv.tomato DC3000 appears to be repressed under high iron concentration conditions (Jones and Wildermuth, 2011).While virulence control by iron has been well illustrated for the Fur system in P. aeruginosa, the Fur system is a negative regulator, indicating that the system represses the uptake of iron when iron is rich (Lamont et al., 2002).Furthermore, expression analyses in P. syringae pv.tomato DC3000 cultures indicates that high iron [50 M iron(III) citrate] both represses high-affinity iron-scavenging system expression and induces expression of the type III secretion system and virulence genes in culture (Jones and Wildermuth, 2011).From our data it seems that hem was suppressed in the high iron concentrations at 6 mM and in the plant cells which assumed is an also high iron concentration.Therefore, hem mutant shows the disease severity as a wildtype when inject the cells directly to soybean plant.Other pathogens that colonize similar plant environments (that is, the leaf apoplast and vasculature) might be expected to be similarly.The addition of bean leaf apoplastic fluid to Pseudomonas syringae pv.phaseolicola NPS3121 grown in minimal medium resulted in the expression of virulence genes and the repression of highaffinity iron import systems (Hernández-Morales, 2009).It was expected that abundant iron would repress highaffinity iron scavenging, but the induction of virulence genes was surprising.In a follow-up study, Kim et al. (2009Kim et al. ( , 2010) ) found that expression of virulence factors of Pseudomonas syringae pv.tomato DC3000 in hrpinducing minimal medium was limited by iron availability and that higher iron to well above 10 M continued to induce higher virulence gene expression.Indeed, we observed that the hem mutant did not affect type III genes expression under hrp-inducing minimal medium, this result suggesting that type III secretion system may be involved via a mechanism independent of the hem gene.For growth in vitro, the hem mutant exhibits an increase in sensitivity to iron when grown in the high iron media, compared to the wildtype.The growth effect of hem mutant under high iron concentration might be due to iron catalyzes the Fenton reaction as in Fur and Zur systems.This growth defect phenotype also found in the fur and zur mutants in X. campestris pv.campestris and X. oryzae pv.oryzae respectively (Jittawuttipoka et al., 2010;Tang et al., 2005;Yang et al., 2007).The zur mutant was also exhibited an increase in sensitivity to zinc or iron when grown in the high zinc or iron media compared to the wildtype in X. oryzae pv.oryzae (Yang et al., 2007).In case of P. syringae pv.tomato DC3000, the iron-rich condition is around 200 M and iron toxicity begins at over 400 M.The toxicity was expected because iron catalyzes the Fenton reaction, producing the highly reactive hydroxyl radical, that result in reduced aerobic growth (Andrews et al., 2003).Moreover, the haem-uptake gene cluster in Vibrio fischeri is also regulated by Fur and contributes to symbiotic (Septer et al., 2011).
The successful establishment of a pathogen within a specific niche requires the ability of the pathogen to sense the specific environmental conditions of the host and to regulate the expression of virulence genes accordingly (Mekalanos, 1992).It is interesting to speculate that in response to hemin limitation, X. axonopodis pv.glycines is capable of turning on the expression of several factors which appear to be involved in the virulence potential of this organism.In this study report that hem was affected to the EPS production, biofilm formation, attachment, and motility but did not for extracellular enzymes production.This inference is consistent with the observations of hemin uptake system to enhanced expression of several putative virulence factors by Streptococcus pneumoniae and Porphyromonas gingivalis in mammalian hosts (Tai et al., 1993;Genco et al., 1995).Similar to the zur gene, the X. oryzae pv.oryzae zur mutant decreased the production of EPS and virulence on rice (Yang et al., 2007).
Previous report indicated that the existence of significant amounts of iron in corn seeds affected in bacterial adhesion and host colonization processes (Jacobs and Walker, 1977).The effect of hemin system to a role in EPS synthesis, and reduction in biofilm formation has also been observed in hemin transport protein mutants in Yersinia (Jarrett et al., 2004).
Importantly, we also found that the hem mutant was deficient in adhesion to both abiotic surfaces and soybean leaf surfaces.A clinical study has shown that iron depletion alters the cell surface property of pathogenic bacteria and lowers their attachment to sur-faces (Harjai et al., 1996).It is therefore possible that iron limi-tation reduces the ability of the hem mutant to attach to soybean leave leading to a lower colonization rate.In contrast with P. gingivalis, the decreased transport of hemin by P. gingivalis results in the increased expression of hemolytic and trypsin-like protease activities that may contribute to the enhanced invasiveness exhibited in the mouse subcutaneous chamber model (Genco et al., 1995).Moreover, we also found that the swimming ability of the hem mutant was reduced.For marine bacteria that specialize in living on particles and aggregates, the population swimming speed of marine bacteria were significantly reduced in no-iron treatment.This reduction in population swimming speeds resulted in lower diffusivity and subsequently a lower colonization rate (Tang and Grossart, 2007).
We were surprised to find that the level of extracellular enzyme productions was not differed in the hem mutant compared with the Xag wildtype.Since changes in quorum sensing are associated with varying iron levels and quorum sensing of Xag controls a variety of traits including extracellular enzymes production, EPS production, motility, and biofilm formation that contribute to the virulence and epiphytic fitness (Thowthampitak et al., 2008).In Xanthomonas campestris pv.campestris, the strain deficient in exbD2, which encodes a component of its unusual elaborate TonB system, had impaired pectate lyase activity and caused no visible symptoms for defense on the non-host plant pepper (Vorhölter et al., 2012).It seems possible that extracellular enzyme productions of Xag may be regulated by other iron uptake pathway.Indeed, disruption of hem impairs the epiphytic fitness of Xag, as observed by a significant decrease in the population size of the mutants on soybean leaves compared to wild-type.It might thus be expected that, hem of X. axonopodis pv.glycines strongly effects to the ability of bacteria to EPS production, biofilm formation, swimming motility and thus lead to advantage for survival and colonization on leave surface, dispersal throughout the soybean plant, and start the new cycle of pustule disease.

Figure 1 .
Figure 1.Overlap extension PCR was used to create constructs in the suicide-delivery vector pTOK2 to create site-directed mutant of hem gene in Xanthomonas axonopodis pv.glycines 12-2 by recombination (A).A hem-complementary strain, the 0.8-kb sequence of hem containing the native promoter was amplified and ligated into the multiple cloning site of vector pBBR1MCS-5(B).

Figure 2 .
Figure2.The growth of Xanthomonas axonopodis pv.glycines strains under different FeCl3 concentration.Culture (10 l, cell density adjusted to about 10 8 cells/ml) of each strains was inoculated into 5 ml of NYGB supplemented with different concentration of Fe 3+ and were incubated at 28°C in shaker.WT = wildtype, hem -=hem mutant, and hem + = complemented hem.The cell density was measured spectrometrically at 600 nm after incubation for 24 h.Bars represent standard error of the means.

Figure 3 .
Figure 3. Virulence testing of the Xanthomonas axonopodis pv.glycines strains when bacterial cells were sprayed on soybean (A) and were injected into soybean cotyledons (B).WT = wildtype, hem -= hem mutant and hem -= hem complementary strain.

Figure 4 .
Figure 4. Relative abundance of transcripts of hem gene in Xanthomonas axonopodis pv.glycines wildtype (WT) and hem mutant (hem -) grown in culture medium compared with wildtype in soybean plant as determined by real-time qRT-PCR.Vertical bars represent the standard error of mean ratio.

Figure 9 .
Figure9.The motility zone of colonies grown in swimming and swarming plates with 0.25% and 0.4% agar respectively was measured at 2 days.WT = wildtype, hem -= hem mutant and hem -= hem complementary strain.Each data point is an average of 4 independent experiments, error bars indicate the standard error.The results are representative of four independent experiments.

Strain Mean of lesions per plant a Relative virulence (%)
a Data shown are the averages ± standard deviations.