Xanthomonas oryzae pv. oryzicola ( Xoc ) causes a serious economically important disease of rice known as Bacterial Leaf Streak (BLS). The bacterium gains entry through stomata or wounds and grows in the inter‐cellular space of the parenchyma (Niňo‐Liu et al ., 2006 ). Very little is known about the virulence function of Xoc which contributes to its growth and survival inside rice host. Recent comparative and functional genomic analysis of Xoc has revealed conserved functions associated with virulence in other plant pathogens, such as Type III secretion system; two component regulatory system; LPS, Type IV pili for twitching motility and components of DSF/RpfF cell–cell signalling (Wang et al ., 2007 ; Bogdanove et al ., 2011 ). The aim of this study was to understand how DSF/RpfF mediated cell–cell signalling promotes in planta growth and virulence of this important phytopathogen. To understand the role of DSF in Xoc virulence, we have characterized the DSF deficient rpfF deletion mutant strain of Xoc which exhibited in planta growth and virulence deficiency. Our study revealed that DSF promotes in planta growth of Xoc by positively regulating functions required for ferric (Fe 3+ ) iron uptake, which is critical for virulence and growth of this pathogen inside rice host. Our study also indicates that closely related pathovars of X . oryzae ( Xoc and Xoo ), which exhibit different tissue specificity and lifestyle (rice xylem vessel vs. parenchyma), may employ alternate iron uptake strategy to utilize different forms of iron (ferrous or ferric form of iron) based on its availability and contrasting lifestyle inside the host.

In several members of the genus Xanthomonas and its close relative Xylella , which constitute economically important plant pathogens, cell–cell signalling is mediated by the production and perception of fatty acid signalling molecules ( cis ‐11‐methyl‐2‐dodecenoic acid) known as diffusible signal factor (DSF) (Chatterjee et al ., 2008b ; Bűttner and Bonas, 2010 ; Deng et al ., 2011 ). In Burkholderia cenocepacia , the DSF synthase RpfF has been reported to encode a promiscuous synthase with duel activity; a bifunctional crotonase having both dehydratase and thioesterase activities (Bi et al ., 2012 ). Although DSF/RpfF is conserved among different members of the genus Xanthomonas and its close relative, different members of this genus exhibit atypical regulation of virulence associated functions controlled by DSF ( Chatterjee et al ., 2008ab ; Rai et al ., 2012 ). In X . campestris pv. campestris ( Xcc ), DSF/RpfF system positively regulates production of extracellular polysaccharide, several Type II effectors and negatively regulate biofilm formation in a density depended manner (He et al ., 2006 ). In contrast to Xcc , studies in X . oryzae pv. oryzae ( Xoo , a vascular pathogen of rice) and Xylella fastidiosa (pathogen of grape) has indicated that DSF/RpfF system regulates virulence associated traits in a contrasting fashion, wherein DSF/RpfF promotes biofilm formation and negatively regulate functions required for planktonic lifestyle (Chatterjee et al ., 2008a ; Rai et al ., 2012 ). Recent studies in the model Xanthomonas phytopathogen, Xcc has indicated that different regulatory components of the DSF/RpfF system which includes two component hybrid sensor and response regulator (RpfC‐RpfG) exhibits atypical or divergent regulatory patterns, indicating the complexity of the DSF signalling in regulating virulence associated functions (An et al ., 2013 ). In X . fastidiosa it has been demonstrated that DSF deficient rpfF mutants have contrasting phenotype as oppose to rpfC (putative DSF sensor; Chatterjee et al ., 2008a ; Beaulieu et al ., 2013 ). Interestingly, DSF synthase RpfF protein of X . fastidiosa plays an unusual role in DSF perception which is independent to its catalytic activity (Ionescu et al ., 2013 ). These studies clearly indicate that DSF/RpfF system is even more complex than previously anticipated and support the idea that closely related Xanthomonads utilizes DSF mediated altered regulation and mechanism as an adaptation to suite specific lifestyle of these bacteria (Chatterjee et al ., 2008b ; Rai et al ., 2012 ).

In several bacteria, cell‐to‐cell signalling is mediated by the production and sensing of small diffusible signalling molecules, which plays an important role in the co‐ordination of social behaviour and adaptation to different environmental conditions (Bodman et al ., 2003 ; Ng and Bassler, 2009 ). Cell‐to‐cell signalling in bacteria co‐ordinates multiple community behaviour in a density depended fashion such as production of secreted virulence factors (cell wall hydrolyzing enzymes), functions required for attachment, motility and biofilm formation (Parsek and Greenberg, 2005 ; Waters and Bassler, 2005 ; Ng and Bassler, 2009 ). Bacteria produces diverse type of signalling molecules which differ widely in their chemical nature as well as in their mode of action: signal perception and signal transduction (Williams et al ., 2007 ; Ng and Bassler, 2009 ). In general, the most widely studied cell‐to‐cell signalling system in gram negative bacteria is mediated by the production and sensing of Acyl‐Homoserine lactone family of signalling molecules (Fuqua et al ., 2001 ).

Exogenous iron promotes growth of Δ rpf F mutant of Xanthomonas oryzae pv. oryzicola in rice leaves . Wild‐type (BXOR1), Δrpf F mutant and Δ rpf F mutant harbouring the plasmid‐borne wild‐type allele (pSC9) strains were inoculated on detached rice leaves maintained in water containing 1 μg/ml of Benzyl amino purine (BAP), with (50 μm FeCl 3 ) or without iron supplementation. Bacterial growth was determined after 0, 24 and 48 h post inoculation. Values presented are means ± standard deviation of log (cfu/cm 2 ) from three independent experiments (three leaves each from three independent experiments). The asterisks indicate P < 0.05 (T‐test) significant difference between the data obtained for the Δ rpf F mutant strain and those obtained from wild‐type BXOR1 strain and Δ rpf F mutant harbouring the plasmid‐borne wild‐type rpf F allele.

To determine whether iron supplementation would rescue the growth defect of Xoc ΔrpfF mutant inside the rice leaves, detached rice leaf assay was done as described previously (Chatterjee and Sonti, 2002 ). In this experiment detached rice leaves were maintained in flask with or without iron supplementation (see Experimental procedures ; Fig. S10 ). These detached rice leaves were inoculated with different strains of Xoc , and bacterial growth within the leaves was estimated after 24 and 48 h post inoculation. In planta growth assay indicated that Δ rpfF mutant exhibited at least 10‐fold less growth compared to either the wild‐type BXOR1 or Δ rpfF mutant strain harbouring the plasmid‐borne wild‐type rpfF allele in rice leaves dipped in medium without any iron supplementation after 24 and 48 h post inoculation, respectively (Fig. 9 ). The growth of the Δ rpfF mutant strain increased substantially in rice leaves supplemented with exogenous iron compared to leaves dipped without any iron supplementation. In contrast, iron supplementation in rice leaves did not significantly affect the growth of either the wild‐type BXOR1 strain or the Δ rpfF (pSC9) in rice leaves dipped in medium either with or without any iron supplementation (Fig. 9 ).

It has been reported that many bacteria utilize extracellular or cell‐associated ferric reductase activity to reduce Fe 3+ to Fe 2+ form of iron which may contribute to the availability of free iron from the environment (Schröder et al ., 2003 ). To examine whether any altered ferric reductase activity may contribute to the reduced capacity of DSF deficient Δ rpfF mutant to utilize Fe 3+ form of iron, we performed ferric reductase activity assay using the specific Fe 2+ chelator ferrozine, which forms a magenta‐coloured Fe 2+ ‐ferrozine complex (Velayudhan et al ., 2000 ). Ferric reductase assay indicate that Xoc exhibits extracytoplasmic ferric reduction activity ( Fig. S9B ). Moreover, there was no significant difference in the ferric reductase activity exhibited by the wild‐type BXOR1 strain, Δ rpfF mutant and Δ rpfF mutant harbouring the complementing plasmid ( Fig. S9B ). Under similar assay condition, no spontaneous reduction of ferric iron was observed in the cell free media which was used as a control.

Δ rpf F mutant exhibits a defect in Fe 3+ uptake. Incorporation of radiolabelled Fe 3+ by wild‐type BXOR1 strain (closed circles), the Δ rpf F mutant (closed squares) and Δ rpf F (pSC9), Δ rpf F mutant harbouring the complementing plasmid (closed triangles). Transport was initiated by the addition of either (A) 0.4 μM 55 FeCl 3 or (B) 0.4 μm of 1:1 ratio of 55 FeCl 3 and vibrioferrin, to cell suspensions of different strains of X oc grown under low‐iron conditions. The data presented represent means and standard deviation from three separate experiments (each with three replicates). The asterisks indicate P < 0.05 (T‐test) significant difference between the data obtained for the Δ rpf F mutant strain and those obtained from wild‐type BXOR1 strain and Δ rpf F (pSC9).

To confirm that DSF in Xoc promotes iron uptake/ metabolism, 55 Fe 2+ and 55 Fe 3+ uptake assays were performed with wild‐type BXOR1 strain, Δ rpfF mutant and the Δ rpfF mutant harbouring the plasmid‐borne wild‐type rpfF allele. For Fe 3+ uptake assay 55 FeCl 3 was used. For Fe 2+ uptake assay, ascorbate was used to reduce the ferric (Fe 3+ ) to ferrous (Fe 2+ ) form of iron by diluting the 55 FeCl 3 stock in 1M sodium ascorbate as described previously (Velayudhan et al ., 2000 ; see Experimental procedures ). For uptake assays different strains of Xoc were grown in low‐iron media (PS + 50 μM 2′2′‐bipyridyl) for 24 h and uptake assay was done in PS media treated with chelex‐100 as described in Experimental procedures . The total amount of radiolabelled Fe 3+ incorporated into the Δ rpfF mutant was significantly less than that incorporated into the wild‐type BXOR1 strain and the Δ rpfF (pSC9) over the 15 min time‐course of the experiment (Fig. 8 A). We also performed Fe 3+ uptake assay with 1:1 ratio of 55 FeCl 3 and vibrioferrin (Fig. 8 B). In the presence of vibrioferrin, the total amount of radiolabelled Fe 3+ incorporated into the Δ rpfF mutant was increased and was similar to that incorporated into the wild‐type BXOR1 strain and the Δ rpfF (pSC9) (Fig. 8 B).

The Δ rpf F mutant of X oc exhibit reduced production of vibrioferrin. BXOR1, Δ rpf F and Δ rpf F (pSC9) were grown under low‐iron condition with 50 μM of 2,2′ dipyridyl (DP) and grown to OD 600 1.Siderophore was purified from culture supernatant by Amberlite XAD‐16 resin column chromatography. For estimating vibrioferrin concentration, cell normalized siderophore fraction was dissolved in methanol and analyzed by HPLC (see supporting Experimental procedures ). Vibrioferrin was detected at 300 nm and the concentration was determined based on their peak area (mUA × min) and calculated from standard curves generated for known concentration of purified vibrioferrin. Siderophore concentration of each strain is represented as μg/ 10 8 CFU and error bars represents ± S.D. of three independent experiments. The asterisk indicates P < 0.05 (T‐test) significant difference between the data obtained for the Δ rpf F mutant strain and those obtained from the wild‐type BXOR1 strain and Δ rpf F (pSC9).

In order to quantitate siderophore production, we isolated siderophore from the cell free culture supernatant of wild‐type BXOR1 strain, Δ rpfF and Δ rpfF (pSC9) by Amberlite XAD‐16 resin column chromatography as described in the supporting Experimental procedures . Siderophore containing fractions were analyzed by HPLC (see supporting Experimental procedures ; Fig. S8 ), and compared with the elution profile of standard purified vibrioferrin (Fujita et al ., 2011 ). HPLC based quantitative analysis indicated that the Δ rpfF produced at least fourfold less vibrioferrin than that produced by the wild‐type BXOR1 strain and the Δ rpfF (pSC9) (Fig. 7 ).

To further understand the contribution of ferric uptake in Xoc virulence and growth under low iron conditions, we created a nonpolar mutation in the Xoc xanthomonas siderophore synthesis gene, xssA (XOC_3387) ( Table S1 ; see Experimental procedures ). Growth assays under low‐iron conditions; exogenous supplementation with Fe 2+ and Fe 3+ forms of iron; and virulence assay on rice leaves indicated that the xssA ::pK18mob mutant of Xoc phenocopies the Δ rpfF mutant (Fig. 6 ). Overall, these results indicated that DSF regulated ferric iron uptake in Xoc is required for growth under low‐iron condition and optimum virulence.

Relative quantification of expression of siderophore biosynthesis ( xss A ; xss B ) and TonB‐dependent receptor (XOC_1096) of X oc regulated by DSF and low‐iron conditions by real‐time RT‐PCR. Different strains of X oc ; BXOR1 (wild type), Δ rpf F and Δ rpf F (pSC9) were grown to OD 600 1.2 in PS medium, PS plus 50 μM 2′2,dipyridyl (DP) alone or supplemented with 30 μM FeSO 4 . Amount of RNA relative to that in the wild‐type BXOR1 cells grown in PS media is equal to 1.0 and is normalized for cellular abundance by using 16S ribosomal RNA as an endogenous control. Standard errors were calculated based on at least three independent experiments.

It has been reported that Xanthomonas siderophore synthesis and uptake ( xss and xsu ) locus is homologus to the Vibrio parahaemolyticus pvs locus, involved in the synthesis and uptake of vibrioferrin, an α‐hydroxy carboxylate‐type of siderophore and expression of xss and xsu genes are induced under iron‐limiting conditions (Pandey and Sonti, 2010 ). To investigate the role of DSF and iron in the expression of ferric uptake genes, we performed real‐time RT‐PCR analysis of RNA isolated from cultures of BXOR1 (wild type), Δ rpfF and Δ rpfF (pSC9) grown under iron‐deprived and iron‐replete conditions. Real‐time RT‐PCR analysis indicated that the expression of genes involved in siderophore biosynthesis, xssA and xssB , were highly induced under low‐iron conditions (Fig. 5 ). In addition, supplementation with 30 μM FeSO 4 in low‐iron medium (PS plus 50 μM 2, 2′‐dipyridyl) suppressed the expression of siderophore biosynthetic genes (Fig. 5 ). Under low‐iron conditions, expression of xssA and xssB were five‐ and threefold lower in the Δ rpfF mutant compared to the wild‐type BXOR1 strain, respectively (Fig. 5 ). Expression of a TonB‐dependent receptor (XOC_1096) was approximately 8‐ to 10‐fold lower in Δ rpfF compared to the wild‐type BXOR1 strain grown either in PS or in low‐iron medium (Fig. 5 ). The expression of an xsuA :: gusA transcriptional fusion in the wild‐type BXOR1 strain background was analyzed under rich PS media and iron‐limiting conditions (See supporting Experimental procedures ). High levels of β‐glucuronidase activities were observed under low‐iron conditions (PS + 50 μM DP) ( Fig. S7 ). In contrast, a very low basal level of expression was observed for the xsuA :: gusA fusion during growth in either rich PS medium ( Fig. S7 ). Furthermore, GUS expression in extracts of rice leaves infected with the xsuA :: gusA ‐BXOR1 strain indicated that the xsu operon is expressed during in planta growth of Xoc ( Fig. S7 ).

We also confirmed the pattern of expression of 11 DSF‐regulated iron uptake/metabolism genes in the Xoc wild‐type BXOR1, Δ rpfF , and Δ rpfF (pSC9) strains grown under low‐iron condition by real‐time Reverse Transcriptase‐Polymerase Chain Reaction (RT‐PCR) to validate the microarray results (Fig. 4 ; Table S5 ). Interestingly, expression analysis of DSF‐regulated genes involved in iron uptake/metabolism indicated that, in general, majority of them are involved in the ferric (Fe 3+ ) iron uptake (Fig. 4 ; Table S5 ). For example: Several putative TonB‐dependent receptors (XOC_0143, XOC_1096, XOC_1946, XOC_4682); ferric citrate transport ( fecA ; XOC_3386) and functions involved in Xanthomonas siderophore synthesis and uptake ( xss and xsu ; XOC_3387, XOC_3388, XOC_3389, XOC_3390, XOC_3391) were down‐regulated in the Δ rpfF mutant (Fig. 4 ; Table S5 ).

DSF‐regulated genes involved in iron uptake and metabolism based on expression analysis by microarray and real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR). The y‐axis represents fold change in expression (log 2 ). Microarray expression is represented as geomean ratio [* Fold changes (log 2 )] of Δ rpf F versus either the wild‐type strain (BXOR1) or Δ rpf F(pSC9). Genes that were significantly up‐regulated by 0.6 or more or down‐regulated by −0.6 or less fold were identified. The asterisks indicate differentially expressed genes based on microarray expression analysis, which were also validated by real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR). For RT‐PCR, data were normalized to an internal 16s rRNA control, and the relative changes in transcriptional level were calculated as a ratio of transcript levels of Δ rpf F versus the wild‐type BXOR1 strain using log 2 of fold difference method. Data represent the means of three independent experiments ± SEM.

In an attempt to gain better insights into DSF‐mediated regulation of iron uptake/ metabolism functions in Xoc, we used microarray‐based gene expression profiling using an Agilent platform, as described below, using a custom‐made 8‐by‐15‐k array based on the sequenced strain of Xoc BLS256 (Bogdanove et al ., 2011 ). RNA was isolated from cells grown to OD 600 1.2 in PS plus 50 μM 2, 2′‐dipyridyl (low‐iron conditions). Two replicates each for the wild‐type BXOR1 strain, Δ rpfF mutant and Δ rpfF (pSC9) were used. Genes which were significantly up‐regulated by 0.6 or more (equivalent to fold change of 1.5) or down regulated by −0.6 or less‐fold (log 2 ratio of expression) were considered for further analysis. T‐test p‐value was calculated using volcano Plot (see Experimental procedures ; Rai et al ., 2012 ). Genes were classified based on functional category. By comparing the expression of genes in Δ rpfF mutant of Xoc with either the wild‐type BXOR1 strain or the Δ rpfF (pSC9), we identified 188 genes that are positively regulated by DSF and 145 genes that are negatively regulated, respectively ( Fig. S5 and S6 ; Table S4 ). Based on their annotation in the genome sequence of Xoc BLS256 (Bogdanove et al ., 2011 ), DSF‐regulated genes were grouped into 13 major functional classes which include: I) extracellular cell wall hydrolyzing enzymes, II) LPS and EPS bio synthesis, III) multidrug resistance and detoxification, IV) chemotaxis, V) hypersensitive reaction and pathogenicity system, VI) nucleic acids and transcription regulators, VII) iron uptake VIII) protein and amino acid metabolism, aerobic and anaerobic respiration, IX) small nucleotide binding regulators, X) membrane components and transporters, XI) fatty acid metabolism, XII) two component systems and regulators, and XIII) hypothetical protein. ( Fig. S6 ; Table S4 ). Expression analysis indicated that 6.9 percent (13/188) of genes which are positively regulated by DSF are involved in iron uptake and metabolism (Fig. 4 ; Table S4 and S5 ). In contrast, only 2 (XOC_3378 and XOC_3709) out of 145 genes that are negatively regulated by DSF are involved in iron uptake and metabolism (Fig. 4 ; Table S4 and S5 ).

To further confirm the intracellular iron content, cells of wild type and ΔrpfF and ΔrpfF (pSC9) were analyzed quantitatively by Inductively Coupled Plasma‐Optical Emission Spectrometry (ICP‐OES; see Experimental procedures ) for elemental iron. The ΔrpfF mutant strain contained threefold less cellular iron levels compared to the wild‐type BXOR1 strain when cultured in PS medium (Table 1 ). Additionally, growth in the presence of 2, 2′‐dipyridyl caused an overall reduction in cellular iron levels in the wild type (BXOR1), ΔrpfF and ΔrpfF (pSC9). However, the iron content of the ΔrpfF mutant was still threefold lower than that of either the wild‐type BXOR1 strain or ΔrpfF (pSC9) (Table 1 ).

The quinone antibiotic streptonigrin (SNG) has been reported to act as bactericidal by promoting formation of oxygen radicals and its bactericidal activity is directly correlated to intracellular iron content (Cohen et al ., 1987 ). It has been shown that bacterial cell with higher intracellular iron content are hypersensitive to SNG (Yeowell and White, 1982 ; Schmitt, 1997 ). Hence, to further assess the iron levels in the different Xoc strains, we performed SNG sensitivity assay. Growth assay on plates with different concentration of SNG and in broth cultures suggested that the ΔrpfF mutant can tolerate higher levels of SNG compared to the wild‐type BXOR1 strain, which is indicative of lower intracellular free iron inside the mutant (Fig. 3 ). The ΔrpfF (pSC9) exhibited less tolerance to SNG, similar to that exhibited by the wild‐type BXOR1 strain (Fig. 3 ). This result indicated that the Δ rpfF mutant has a lower level of free intracellular iron.

It is generally assumed that nutrient and iron uptake/metabolism plays an important role in the growth and survival of bacterial pathogens inside their hosts (Franza and Expert, 2010 ; Rohmer et al ., 2011 ; Expert et al ., 2012 ; Cassat and Skaar, 2013 ). To test if the virulence deficiency exhibited by the Δ rpfF mutant strain is due to a defect in nutrient metabolism or uptake, we compared the growth of the wild‐type BXORI, Δ rpfF and Δ rpfF (pSC9) strain on rich peptone sucrose agar (PSA), minimal medium (MM9; Kelemu and Leach, 1990 ) and low‐iron medium (PSA medium supplemented with iron specific chelator 2,2′‐dipyridyl). The ΔrpfF mutant of Xoc did not exhibit any growth defect compared to the wild‐type BXOR1 strain on either rich (PSA) or minimal medium, respectively ( Fig. S4 ). However, ΔrpfF mutant of Xoc exhibited reduced growth on PSA plates containing iron specific chelator 2,2′‐dipyridyl ( Fig. S4A ). In a control experiment, the addition of DSF isolated from the cell‐free culture supernatant of either the wild‐type strain or a siderophore deficient xssA ::pK18mob mutant strain complemented the in vitro growth deficiency exhibited by the ΔrpfF mutant strain under low‐iron condition ( Fig. S4B ). In order to gain better insight into the role of DSF in iron acquisition, we compared the growth of different strains of Xoc in rich Peptone Sucrose (PS) broth in the presence of Fe 2+ specific chelator 2,2′‐dipyridyl (Fig. 2 ; Table S2 ) . In PS medium alone, the growth rates of wild‐type BXOR1 and ΔrpfF mutant were indistinguishable. Compared to growth in the PS medium (doubling time of approximately 3.3 h), the wild‐type BXOR1 strain exhibited growth deficiency (doubling time 5.8 h) in PS plus 100 μM 2,2′‐dipyridyl. In PS plus 100 μM 2,2′‐dipyridyl, the growth rate of ΔrpfF was substantially less than that of BXOR1 strain (Fig. 2 ; Table S2 ). The severe growth defect exhibited by the ΔrpfF mutant strain under iron‐limiting conditions could be rescued by either supplementation of 20 μM FeSO 4 (Fe 2+ ) or by complementation of ΔrpfF mutant with the wild‐type rpfF allele (Fig. 2 ; Table S2 ). Taken together, these data suggested that the significant growth defects exhibited by the ΔrpfF mutant strain under low‐iron conditions may be due to defect in its capacity to sequester sufficient iron. Interestingly, supplementation of 20 μM FeCl 3 (Fe 3+ ) could partially rescue the growth defect exhibited by the ΔrpfF strain grown under low‐iron conditions (Fig. 2 ; Table S2 ). We then examined the growth of BXOR1, ΔrpfF and ΔrpfF (pSC9) in low‐iron medium supplemented with different concentration of both Fe 3+ and Fe 2+ form of iron (Fig. 2 ; Table S2 ; Table S3 ). Iron supplementation experiments indicated that at higher concentrations, both FeSO 4 (Fe 2+ ) or FeCl 3 (Fe 3+ ) was equally efficient in restoring the growth of either the wild‐type BXOR1 strain or the ΔrpfF (pSC9) (Fig. 2 ; Table S2 and S3 ). However, at lower concentrations (20 μM or less), Fe 2+ was better than Fe 3+ in rescuing the growth defect exhibited by the ΔrpfF mutant (Fig. 2 ; Tables S2 and S3 ). These results indicated that the DSF in Xoc may be involved in enabling ferric iron (Fe 3+ ) uptake or metabolism.

C. Lesion lengths on rice leaves caused by different strains of X oc . For needleless syringe inoculation experiment, 15 leaves were inoculated each in three independent experiments and the values are presented as mean lesion length ± standard deviation. The asterisk indicates P < 0.05 (T‐test) significant difference between the data obtained for the Δ rpf F mutant strain and those obtained from wild‐type BXOR1 strain and Δ rpf F mutant harbouring the plasmid‐borne wild‐type rpf F allele. (D) In planta growth of different strains of Xoc; BXOR1 (wild type), Δrpf F (DSF ‐ ) and ΔrpfF (pSC9) (DSF + ). Bacterial populations were measured at the days indicated post inoculation (day 0). Values presented are means ± standard deviation of log (cfu per cm 2 ) from three independent experiments (three leaves each from three independent experiments).

Xoc causes bacterial leaf streak (BLS) disease with streaks like symptoms on the rice leaf (Fig. 1 and S3 ). The strains were inoculated by syringe infiltration on leaves of 6‐week‐old rice plants and assayed for virulence by examining the water soaking and streak like symptoms. Approximately 4 days post inoculation, the wild‐type BXOR1 exhibited typical water soaking symptom (Fig. 1 A). In comparison to the wild‐type BXOR1 strain, ΔrpfF mutant always exhibited less vigorous water soaking symptom (Fig. 1 A; 4 days post inoculation) as well as lesion symptom (Fig. 1 B; 10 days post inoculation). The lesions (streak symptom) caused by wild‐type BXOR1 strain is approximately 1.5 cm long whereas of ΔrpfF (deletion mutant) is 0.5 cm long 10 days after inoculation by syringe infiltration (Fig. 1 C). We performed virulence assay by wound inoculation. In this assay, 20 μl of bacterial suspension (∼1 × 10 8 cfu per ml) are placed on the expanded leaves and prick inoculated with a hypodermic syringe needle (see Experimental procedures ). The lesions (streak symptom) caused by wild‐type BXOR1 strain is approximately 2.8 cm long 14 days after inoculation by the leaf prick method ( Fig. S3 ). In contrast, the lesions caused by ΔrpfF (deletion mutant) are 0.3 cm long 14 days after inoculation ( Fig. S3 ). Compared to wild‐type BXOR1 strain, the ΔrpfF mutant strain always exhibited less vigorous lesion symptoms (streak) on rice leaves, which corresponds to typical BLS caused by Xoc ( Fig. S3A ). Infection efficiency was scored by determining the percentage of infected leaves showing disease lesions 14 days after inoculation. Under the conditions of wound infection (needle prick method), approximately 80% of leaves infected with BXOR1 strain exhibited visible lesions. In contrast, only 25 % of leaves inoculated with the ΔrpfF strain exhibited lesions, which were much reduced than the wild‐type strain ( Fig. S3C ). Complementation of ΔrpfF mutant with the wild‐type rpfF allele (pSC9) rescued the virulence deficiency (Fig. 1 , Fig. S3 ).

Discussion

In this study, we have characterized the role of DSF/RpfF mediated cell–cell signalling in virulence and iron metabolism of rice pathogen Xanthomonas oryzae pv. oryzicola (Xoc). The results of this study have established the following: DSF in Xoc promotes growth under low‐iron conditions; DSF positively regulates functions involved in (Fe3+) iron uptake; DSF deficient ΔrpfF mutant have low intracellular iron content and exhibit defect in Fe3+ iron uptake; and exogenous supplementation of iron promoted in planta growth of ΔrpfF mutant.

It has been shown that in Xanthomonas and other closely related bacteria, such as Xylella fastidiosa (a pathogen of grapes), synthesis of a DSF family of signalling molecules requires rpfF (regulation of pathogenecity factor F), which encodes DSF synthase (RpfF), a bifunctional crotonase having both dehydratase and thioesterase activities (Wang et al., 2004; Bi et al., 2012; Beaulieu et al., 2013; Ionescu et al., 2013). The Xoc DSF synthase RpfF exhibited high homology to the RpfF of other closely related Xanthomonas, including two highly conserved glutamate residues in the catalytic core which are required for DSF synthesis (Fig. S1). Substitution of alanine for these glutamate residues (E141A and E161A) in the Xoc wild‐type rpfF allele resulted in loss of complementation of DSF related phenotypes in the ΔrpfF mutant background (R. Rai and S. Chatterjee unpublished results).

Iron supplementation experiments indicated that the ΔrpfF mutant of Xoc exhibited reduced capacity to utilize Fe3+ form of iron under low‐iron conditions (Fig. 2; Table S2 and S3). Our Iron uptake assays with radiolabelleld 55Fe2+ and 55Fe3+ further demonstrated that that ΔrpfF mutant exhibited significant defect in the ferric (Fe3+) iron uptake but was proficient in ferrous iron transport (Fig. 8). This may be the reason for the fact that Fe2+ form of iron was better than Fe3+, in rescuing the growth defect of the ΔrpfF mutant strain under low‐iron conditions. Although, ΔrpfF mutant exhibited defect in the ferric (Fe3+) iron uptake, supplementation of higher concentration of FeCl 3 (≥ 30 μM) could correct the growth deficiency of the ΔrpfF mutant strain grown under low‐iron conditions (Table S3). It has been reported that many bacterial species exhibit extracellular or cell‐associated ferric reductase activity which reduces Fe3+ to Fe2+ (Schröder et al., 2003). Ferrous form of iron being more soluble is then transported by the ferrous uptake system (feo). In our study, the wild‐type BXOR1 strain of Xoc and ΔrpfF mutant exhibited similar extracytoplasmic ferric reductase activity (Fig. S9B). It is possible that under such conditions (high concentration of FeCl 3 ), sufficient amount of Fe3+ is converted to Fe2+ by the ferric reductase activity, which can then be utilized by the ΔrpfF mutant by the ferrous uptake system. Another possibility is that at higher concentration of iron, low affinity iron transporters could compensate for ferric iron acquisition defect in the ΔrpfF mutant. It has been reported that several bacterial pathogens produce ABC permeases, which promote bacterial growth under low‐iron conditions (Franza and Expert, 2010). For example, in Serratia marcescens, an ABC permease can transport ferric iron without the requirement of a TonB‐dependent outer membrane receptor (Zimmermann et al., 1989; Angerer et al., 1990). It is pertinent to note in this regard that some members of the bacteria belonging to the genus Bradyrhizobium and Rhizobium and several fungi excrete Krebs cycle intermediates such as citric, malic and oxalic acid under iron starvation condition (Carson et al., 1992; Howard, 1999). These organic acids are low affinity iron chelators, and it has been proposed that they promote iron uptake by solubilizing inorganic iron (Howard, 1999; Frawley et al., 2013). In our expression analysis we found that expression of genes encoding fumarate hydratase (XOC_3019; involved in the citric acid cycle) and acetolactate synthase (XOC_3714; involved in 2‐oxo carboxylic acid metabolism) were up‐regulated in ΔrpfF mutant, compared to the wild‐type strain when grown under low‐iron condition (Table S4). We performed High Performance Liquid Chromatography (HPLC) and GC‐MS analysis of organic acid extracted from the cell‐free culture supernatant of different strains of Xoc (Figs S11 and S12). Analysis of cell‐free culture supernatant indicated that the ΔrpfF mutant produced approximately twofold more oxalic acid compared to either the parental wild‐type BXOR1 strain or the ΔrpfF mutant harbouring the plasmid‐borne wild‐type rpfF allele (Table 2; Fig. S11).

Table 2. Estimation of oxalic acid content in the cell free culture supernatant of different strains of Xoc Strains Conc. of oxalic acid (μg/108 cells)a BXOR1 3.057 ± 0.086 ΔrpfF 5.696 ± 0.182* ΔrpfF (pSC9) 3.765 ± 0.897

Our genome‐wide expression analysis indicated that 6.9% (13/188) of genes, which are positively regulated by DSF, are involved in iron uptake and metabolism. Interestingly, the majority of DSF regulated iron uptake genes is involved in ferric iron uptake which includes functions involved in xanthomonas siderophore synthesis and uptake (xss), Ferric‐citrate transporter (fecA) and several TonB‐dependent receptor proteins (Fig. 4; Table S5). Expression analysis and quantification of vibrioferrin siderophore from the cell‐free culture supernatant indicated that the ΔrpfF mutant exhibited at least four‐ to fivefold less production than that produced by the wild‐type BXOR1 strain (Fig. 4; Fig. 7). It is possible that such a modest change in the level of siderophore production may have an effect under iron‐limiting conditions, as certain critical threshold amount of siderphore production may be required to sequester iron from the environment under these conditions. To understand the role of fur (ferric uptake regulator), we performed expression analysis of Xoc fur (XOC_1736) in the wild‐type BXOR1 and ΔrpfF mutant strain grown under iron‐deprived and iron‐replete conditions. In general, the ΔrpfF mutant strain exhibited a higher expression of fur compared to the wild‐type strain (Fig. S13). In Xanthomonas campestris, TonB‐dependent ferric iron acquisition system (exbB, exbD1 and exbD2) has been proposed to play a role in virulence. However, the role of TonB‐dependent machinery in iron uptake and virulence has not been clearly demonstrated in this bacterium (Wiggerich and Pühler, 2000). In Xoo, a xylem limited pathogen of rice, it has been proposed that cell–cell signalling mediated by DSF/RpfF may be required for Fe2+ iron uptake (Chatterjee and Sonti, 2002). Intriguingly, Xoo, mutants deficient in siderophore biosynthesis and utilization were not affected in virulence. Contrastingly, a feoB mutant of Xoo, which is proposed to be involved in Fe2+ iron uptake, exhibited virulence deficiency (Pandey and Sonti, 2010). In contrast, the Xoc siderophore deficient mutant exhibited virulence deficiency (Fig. 6). Furthermore, expression analysis with the xsuA::gusA‐BXOR1 strain indicated that the xsu operon is expressed during in planta growth of Xoc (Fig. S7). This is in contrast to Xoo, wherein, the xsu operon remains turned off during in planta growth of Xoo in rice leaves (Pandey and Sonti, 2010).

In this study, we were unable to obtain a feoB deletion mutant of Xoc, despite several attempts including the addition of ferrous or ferric form of iron in the selection plates. It may be possible that feoB gene is essential in Xoc and may be required for viability of Xoc under laboratory conditions. Although, Xoo and Xoc are closely related members of the genus Xanthomonas which infect rice, they differ remarkably in their mode of infection, colonization and disease symptom. Xoo, a vascular pathogen, gains entry into rice leaf typically through openings known as hydathodes at the tip and leaf margin and multiply inside the xylem vessel (Niňo‐Liu et al., 2006). Xoc, by contrast, is a non‐vascular pathogen of rice, enters the leaf mainly through stomata and colonizes the intercellular space of parenchyma. These different modes of infection and colonization indicate that these species may have to cope with different environmental and ecological conditions during colonization in the host. It is now becoming increasingly evident that pathogenic bacteria employ diverse strategies to sequester and utilize nutrients from the host to colonize different host tissue (Hofreuter et al., 2008; Rohmer et al., 2011). It has been proposed that diversity in metabolic and nutrient acquisition exhibited by several bacterial pathogens may be an adaptive strategy to colonize different host tissue (Hofreuter et al., 2008).

Several bodies of research now clearly indicate that although DSF/RpfF mediated cell–cell signalling is shared by many closely related Xanthomonas sp., there is considerable difference in the pattern or repertoire of DSF regulated traits among closely related members of this genus (Newman et al., 2004; Chatterjee et al., 2008b; Dow, 2008; Rai et al., 2012; Ionescu et al., 2013). The complexity of DSF regulatory mechanisms and atypical regulation of virulence associated traits among closely related members of the genus Xanthomonas has been proposed to play a role in adaptation to suite different lifestyle of these pathogens (Chatterjee et al., 2008b; Rai et al., 2012). Expression analysis of DSF regulated genes in Xoc indicated that several of the virulence associated genes are regulated in a atypical manner. For example, in Xoc, several components of the Type III secretion system and effectors are positively regulated by (down‐regulated in the ΔrpfF mutant). This is in contrast to Xcc, wherein, several components of the type III secretion system are negatively regulated by DSF (Wang et al., 2004; He et al., 2006). Several studies have now indicated that DSF/RpfF mediated signalling and regulatory system is complex and exhibit variation within closely related Xanthomonas. Recent studies in Xcc and Xylella fastidiosa indicate that DSF mediated cell–cell signalling components rpfC and rpfG, regulate different subset of genes and do not phenocopy DSF deficient rpfF mutant (Newman et al., 2004; Chatterjee et al., 2008b; Rai et al., 2012; An et al., 2013; Ionescu et al., 2013). In Xylella fastidiosa, it has been proposed that the DSF synthase RpfF is involved in DSF sensing (Ionescu et al., 2013). Since DSF isolated from either the wild‐type BXOR1 strain or the siderophore deficient mutant could complement the in vitro growth defect exhibited by the ΔrpfF mutant strain under low‐iron condition (Fig. S4), we think the RpfF may not be involved in DSF sensing in this bacterium. Furthermore, in vitro growth analysis under low‐iron conditions indicated that the ΔrpfC and ΔrpfG mutant of Xoc did not phenocopy the ΔrpfF mutant for growth under low‐iron conditions, however, they exhibited reduced virulence and EPS production (Fig. S14). It may be possible that the rpfC and rpfG may be involved in the regulation of virulence associated traits other than iron uptake and metabolism. It is intriguing to note in this regard that in Xcc, a second DSF sensor, RpfS belonging to the PAS domain family of protein was identified which is involved in the regulation of a subset of DSF‐dependent phenotype independent of RpfC (An et al., 2014). However, rpsR homolog is absent in sequenced strains of Xoc and Xoo. It may be possible that the DSF mediated regulation of iron‐uptake or metabolism may be regulated by yet unidentified signalling component/s in Xoc. In our laboratory, we are now trying to identify the components involved in DSF mediated sensing and signal transduction.

In this study, mutation in the Xoc siderophore biosynthetic gene (xssA) caused a defect in the growth under low‐iron conditions, capacity to utilize Fe3+ form of iron and virulence, thus exhibiting a phenocopy of the effect of ΔrpfF mutant (Fig. 6). These results suggested that although closely related phytopathogen (Xoo and Xoc) infect a common host, their requirement of iron and particularly the iron uptake strategy to utilize either Fe2+ or Fe3+ form of iron may vary substantially depending on the lifestyle (colonization in different host tissue; xylem vs. parenchyma tissue). It is intriguing to note that in plants majority of the iron which is transported across vessels is in the form of Fe3+‐citrate complex (Palmer and Guerinot, 2009). In the rice xylem vessel (a niche for colonization of vascular phytopathogen Xoo), sufficient quantity of iron (in the micromolar range) is present in the Fe2+ form (Yokosho et al., 2009). This may be a reason to the fact that siderophore deficient mutants of Xoo are proficient in virulence as they are able to utilize sufficient Fe2+ form of iron which predominates in the xylem sap. In Dickeya dadantii (syn. Erwinia chrysanthemi), a broad host range plant pathogen (which infects plants such as African violet, Arabidopsis), ferric uptake mediated by siderophores is required for virulence and growth in intercellular spaces of the host tissue. It is interesting to note that a feoB mutant of Dickeya dadantii did not exhibit significant defect in pathogenicity on Arabidopsis plants (Franza and Expert, 2010; Expert et al., 2012). It has been proposed that plant pathogens, which colonizes non‐vascular tissue such as apoplast, utilize iron from the storage vesicles (such as plastids), in which iron is mainly present in Ferric (Fe3+) form bound to iron storage protein ferritins or nicotianamine, which is present mainly in the vacuoles (Palmer and Guerinot, 2009; Expert et al., 2012). Chemical speciation studies have indicated that iron is complexed mainly by citrate in both xylem sap and apoplast (López‐Millán et al., 2000). It is possible that Xoc, a colonist of apoplast and intercellular spaces of the parenchyma, utilizes Fe3+ form of iron, and thus, ferric iron uptake/metabolism is critical for its virulence. It is interesting to speculate in this context that the differences in the requirement of different iron uptake systems among phytopathogens and the availability of different forms of iron inside the host may have contributed in the coevolution of host‐pathogen interactions.

In our detached rice leaf assay, ΔrpfF mutant of Xoc exhibited significant enhancement of growth as a response to iron supplementation (Fig. 9). However, iron supplementation did not completely rescue the growth defect under these conditions (Fig. 9). This suggests that in addition to iron uptake functions, DSF/RpfF system may be required for the production of other virulence associated function which may contribute to in planta growth of Xoc. Indeed, our expression analysis indicated that several virulence associated traits were down‐regulated in the ΔrpfF mutant such as functions involved in production of several Type III secretion system components and their effectors; multidrug resistance and detoxification; extracellular polysaccharide and lipopolysaccharide (Fig. S6; Table S4; Bűttner and Bonas, 2010; Ryan et al., 2011). We also performed hypersensitive response and pathogenicity (HRP) assay on non‐host plant tomato with the wild‐type BXOR1, ΔrpfF and ΔrpfF (pSC9) strains. HRP assay indicated that the ΔrpfF mutant strain exhibited reduced HRP symptoms, compared to either the wild‐type BXOR1 strain or the ΔrpfF mutant strain harbouring the plasmid‐borne wild‐type rpfF allele (data not shown).

In summary, we have shown that DSF/RpfF system in Xoc positively regulate functions involved in ferric iron uptake, which promotes in planta growth and virulence. To the best of our knowledge, this is the first direct biochemical evidence which demonstrates active iron uptake system in a member belonging to the Xanthomonas group of pathogens. Our results have indicated that closely related Xanthomonas phytopathogens may employ different iron uptake strategies (ferrous vs. ferric) to utilize different forms of iron depending on their lifestyle inside the host plant.

Our work and that of others have indicated that interplay of cell–cell signalling mediated by diffusible signal molecules and iron availability may play an important role in the coordination of virulence associated traits. In Pseudomonas aeruginosa, it has been reported that quorum sensing and iron availability plays an important role in motility and biofilm formation (Patriquin et al., 2008). Interestingly, iron availability also influences the production of diffusible signal molecules in this opportunistic human pathogen. Recently it has been shown that in Vibrio vulnificus, a halophilic marine pathogen, both iron and quorum sensing regulate the expression of vulnibactin siderophore biosynthetic genes which is required for virulence (Wen et al., 2012). In Pseudomonas syringae pv. syringae (a pathogen of bean plant), it has been shown that iron availability influences quorum sensing regulated traits such as swarming and production of extracellular polysaccharide (Dulla et al., 2010).

Several body of research now indicate that interference of iron uptake/availability may serve as a biocontrol strategy by restricting the growth of pathogenic and non‐beneficial bacterial species (Kloepper et al., 1980; Loper and Buyer, 1991). In a study, Dulla et al., 2010 isolated several plant associated bacteria which can interfere the acyl‐homoserine lactone mediated quorum sensing of P. syringae pv. syringae. Interestingly, it was shown that the inhibitory effect of these plant associated bacteria to interfere with QS of P. syringae pv. syringae was due to their ability to sequester available iron, and therefore low‐iron condition suppressed the quorum sensing regulated virulence traits in this bacterium (Dulla et al., 2010). It is interesting to speculate that in future, detail understanding of the iron uptake/metabolism strategies in Xanthomonas group of plant pathogens may help to develop novel strategies for either selective interference of iron uptake pathways or modulating the host iron levels, which may have applicability in reducing the severity of disease.