BACKGROUND
citrus canker is a disease caused by the phytopathogens xanthomonas citri subsp.
citri, x. fuscans subsp.
aurantifolli and x. alfalfae subsp.
citrumelonis  <cit> .
among the three phytopathogens, the asiatic form , which causes citrus bacterial canker type a, is the most widely spread and severe, attacking all citrus varieties  <cit> .
in brazil, form a is the most important, being found in practically all areas where citrus canker has been detected  <cit> .
similarly to most phytobacterioses, there is no efficient way to control citrus canker.
the only way to eliminate the disease is through the eradication of sick plants, a procedure that brings significant economical losses.
by law, in são paulo state, the main citrus production area in brazil, it is mandated to eliminate all plants around the focus of infection in a  <dig> m radius if the contaminated plants are less than  <dig> % of the planting field and all plants in the planted field if the contaminated plants are more than  <dig> %.
in the latter case, cultivation is then prohibited in the area for the next  <dig> years and there is no payment for lost production to the growers.
considering the importance of the disease worldwide, especially for brazil, a brazilian group sequenced and annotated the complete genome of x. citri subsp.
citri  strain  <dig>  <cit> , which causes citrus canker, and compared it with x. campestris pv.
campestris strain atcc  <dig>  the etiological agent of crucifer black rot.
the citrus subspecies has  <dig>  open reading frames , of which  <dig> % have been assigned function.
in addition, xcc also has two plasmids that have  <dig> genes, and for  <dig>  of them, no role has been proposed.
although the genome of xcc has been characterized and annotated, the inferences made based on in silico analyses require experimental investigation to accurately detect which genes are related to the pathogen-host adaptation process, and which are associated with pathogenesis itself.
therefore, functional genomics studies are necessary to elucidate the machinery required for pathogen installation and proliferation in plants, and the induction of citrus canker symptoms in the host.
from the functional genomic perspective, large scale analysis of mutants by inoculation in host plants allows identification of the genes required for adaptation, pathogenesis and virulence, providing a best understanding of the colonization and infection potential of the bacteria.
in this work, using transposon insertion mutagenesis  <cit> , a library containing  <dig>  mutants of the citrus canker etiological agent x. citri subsp.
citri strain  <dig> was prepared and  <dig>  mutants were analyzed after individual inoculation of host plants.
eight mutants with absent pathogenicity and  <dig> mutants with reduced symptoms in planta, at varying intensities, were identified.
mutated genes were identified by sequencing the total dna of the mutants with altered virulence, allowing the identification of the site of insertion of the transposon used for mutagenesis.
a random selection of these genes was immobilized on a nylon membrane array and expression profiles were analyzed in vivo through nucleic acid hybridization to labeled cdna probes, using targets corresponding to wild xcc strains multiplied in non-infective  or infective conditions .
finally, a comparative genomic analysis of each mutated orf region from xcc with other sequenced xanthomonas genomes allowed the identification of five interesting genomic regions, with two being exclusive to xcc.
the unique characteristics presented by these five regions suggest that they are probably new pathogenicity islands  <cit>  in xcc.
the implications of the proteins encoded by these mutated orfs in host adaptation and colonization processes and citrus canker symptoms induction are discussed.
RESULTS
mutant analysis and in vivo pathogenicity test
to identify and characterize genes involved in pathogenicity and virulence in x. citri subsp.
citri isolate  <dig>  a library of mutants was built through random transposon insertion.
to determine whether transposon insertion affected the ability of xcc to cause disease,  <dig>  mutants of this library were individually inoculated in rangpur lime  plantlets.
assuming the transposon is randomly distributed along the genome in a single-copy manner, the probability of finding one transposon insertion for a certain gene can be calculated by the formula: p =  <dig> - n, where p is the probability of finding one transposon insert within a given gene; x is the length of the gene; g is the length of the genome; and n is the number of transposon inserts present in the population  <cit> .
based on the sequenced genome of citri  <dig>  and considering the main chromosome and two plasmids, the average length of each orf in the xcc genome is  <dig>  bp  <cit>  and the probability of finding one transposon insert for a certain gene is up to 47%.
the mutants identified as having altered pathogenicity in this first round were re-inoculated and re-analyzed, resulting in a final  <dig> mutants showing some symptomatic variation.
the mutants were grouped in five classes according to severity of the major symptoms: total absence of symptoms; watersoaking ; hyperplasia ; necrosis ; and hypersensitive-like response  .
the site of transposon insertion was determined by sequencing for all  <dig> mutants .
in  <dig> mutants the transposon was inserted inside an orf and in four the insertion was at the 5'-end of the orf, probably in the promoter region .
in addition,  <dig> orfs were hit in two independent mutants  and in two cases the same orf was hit in three different mutants , resulting in  <dig> different orfs being hit.
in all cases, mutants having a transposon insertion in the same orf, irrespective of the insertion site, showed the same phenotype as determined by independent evaluations at three different times.
based on the classification proposed by the xcc genome group http://genoma <dig> fcav.unesp.br/xanthomonas, the mutated genes belong to several categories: seven participate in intermediary metabolism; three are classified in the biosynthesis of small molecules; three are involved in macromolecule metabolism; two are cell structure constituents; four participate in another cellular process; two are related to mobile genetic elements; four are involved with pathogenicity, virulence, and adaptation; eight are hypothetical orfs; and two are undefined orfs.
therefore, among the  <dig> mutants there are  <dig> distinct mutated orfs .
to verify that transposon insertion was random, one southern blot analysis was evaluated.
it was possible to estimate that the number of xcc clones with double inserts in their genomes was approximately  <dig> in every  <dig> mutants of the library .
the same analysis was done on all  <dig> mutants and none of them had double inserts.
analysis of the growth curve in planta and in vitro
to analyze the behavior of some mutants in terms of growth in vitro and in planta,  <dig> mutants were randomly selected and analyzed together with the wild type  .
although all mutants were inoculated with the same number of cells, including the wild-type strain, we observed cellular concentration differences after  <dig> days of growth in citrus leaves.
wild type showed cell growth until  <dig> days, and from that point the growth curve in planta remained constant at close to  <dig> cells/cm <dig> of leaf area.
it was possible to group the  <dig> mutants into five distinct patterns based on the numbers of cells per square cm: 1) mutants that showed a low concentration  of cells during the infection period ; 2) mutants that showed an average concentration  of cells during the infection period ; 3) mutants that had high concentrations  of cells during the cellular infection period ; 4) mutants that showed a sigmoid pattern of cell concentration around  <dig> ; and 5) mutants that had an increase in cell number equal to the wild type until the second day and then the concentration was stable  until the 10th day, when it started to fall, reaching close to  <dig> on the last day .
furthermore, the mutant 18d <dig> also presented a sigmoid growth curve, but with a cell concentration above  <dig>
when the same mutants were grown in culture media, it was observed that the cells grew more similarly to the wild type over time.
however, among all mutants tested, the 02h <dig> and 03c <dig> mutants, which in planta had lower cell concentrations , did not cause any symptoms .
intriguingly, both genes are identified as involved with the type iii secretion system , reinforcing its importance in the disease induction process.
gene expression
to show that the mutated genes are related to the altered symptoms of disease and were involved in pathogenesis , the expression of  <dig> orfs was analyzed through nucleic acid hybridization to labeled cdna probes in two situations: cells multiplied in culture medium  and cells multiplied in citrus leaves  .
the results indicated that the orfs xac <dig>  xac <dig>  xac <dig>  xac <dig>  xac <dig>  xac <dig>  xac <dig>  xac <dig>  xac <dig> and xac <dig> are only expressed when xcc is multiplied in vivo; it was not possible to identify expression of these orfs when cells were multiplied in vitro.
a single orf, xac <dig>  showed no significant expression in any of the conditions  .
the two experimental replications showed similar results.
discussion
random mutagenesis through random transposon insertion in vivo in the genome has been widely and successfully used to study several microorganisms, whether pathogens or not  <cit> .
using this technique for pathogenicity and virulence studies of the causal agent of the citrus canker, a library with approximately  <dig>  viable mutants of x. citri subsp.
citri isolate  <dig> was obtained.
through this strategy, the transposon/transposase complex was inserted directly into the cells through electroporation.
southern blot analysis showed that  <dig> %  had a double transposon insertion, which is near that expected from the description accompanying the kit used to obtain mutants, where the rate of double inserts is about 1% of the clones .
after individual inoculation of  <dig>  mutants in rangpur lime  leaves,  <dig> mutants were identified with some alteration in their ability to induce citrus canker symptoms.
the mutated orfs in mutants with altered pathogenicity were identified through dna sequencing.
in this group of mutants there were genes belonging to several functional categories, including genes previously known as being involved in the pathogenesis process, such as the proteins hrpb <dig> and uptc and new genes xac <dig>  xac <dig> and xac <dig>  the symptoms caused by these mutants were also widely variable, and eight of them did not cause disease, which was confirmed by the total absence of symptoms .
in addition, to show that the mutated genes were really involved in the pathogenesis process , the expression of  <dig> orfs was analyzed through nucleic acid hybridization using labeled cdna probes in two situations: cells multiplied in culture medium  and cells multiplied in citrus leaves  .
the analyses of the blots showed that among these genes it was possible to observe the expression of most in planta, which denotes their importance in interaction or adaptation events during the infection process.
however, no ptha mutant was identified, despite xcc having four distinct copies of ptha, two in each plasmid.
it could be that mutation of just one ptha gene does not affect the establishment of xcc in either pathogenicity or symptoms.
swarup and coworkers  <cit>  have shown that mutation in the ptha gene resulted in a complete loss of virulence on citrus, but the amino acid sequence coded by ptha  <cit>  is distinct from all four ptha copies present in xcc  <dig>  <cit> .
we used homologous recombination to disrupt each copy of xcc ptha in order to determine the contribution of each copy to pathogenicity and virulence.
however, this process is not trivial, because we would first have to obtain a null ptha mutant, ie, a mutant with all four copies of this gene mutated.
under these conditions the adaptability of the null mutant could be tested, and, using that mutant, the contribution of each copy of ptha could be evaluated.
another circumstance that may have influenced the absence of identified pthas mutants is the probability of having all the xcc genes mutated in our mutant library, which was only 47%, whereas empirically, it is much easier to hit the main chromosome, due to its size, than the plasmids.
so, the probability of mutating a gene in the plasmid is also very small in relation to the probability of mutating a gene on the main chromosome.
two of the non-virulent mutants carry genes previously described as being necessary for pathogenicity, hrpb <dig>  and hrpxct ; these two genes are part of the hrp  system, which is present in most gram-negative phytopathogenic bacteria, except for agrobacterium, and is part of the ttss  <cit> .
many results indirectly suggest that virulence proteins, also called virulence effectors, are injected by the pathogen directly inside the host cells through a pilus  <cit> .
it is presumed that the effectual proteins stimulate or suppress several cellular functions of the host to benefit pathogen infection  <cit> .
in x. campestris pv.
vesicatoria , the hrp cluster is  <dig> kb and contains six operons, hrpa to hrpf  <cit> .
two regulator genes, hrpg and hrpx, located outside of the larger gene cluster, are responsible for activating the expression of hrp genes in planta and in xvm <dig> synthetic culture media  <cit> .
the mutant for hrpb <dig> in xcv was not able to cause disease in susceptible pepper plants or the hypersensitive reaction  in pepper plants carrying the respective compatible r gene, in the presence of avr in the xcv isolate used in the study  <cit> .
subsequent studies confirmed that this protein, hrpb <dig>  was not secreted; in other words, it is a protein that acts in the bacterial cell.
in the same study, hrpb <dig> remained in the soluble protein extract while avrbs <dig> was secreted by the wild isolate, which was not observed in a hrpb <dig> defective mutant.
the gene hrpxv  was characterized and its function was determined.
the amino acid sequence deduced indicated similarity with proteins of the arac family, which act in the regulation of gene expression.
mutations at position  <dig>  of that gene stopped the resulting mutant from inducing disease symptoms in susceptible pepper and tomato plants and hr in resistant plants.
complementation with fragments of that gene showed that only  <dig> bp after the initiator codon is enough to produce a functional polypeptide.
the cell concentration of hrpx mutants in planta revealed that the mutant had  <dig> times less bacteria than the wild type genotype  <cit> .
these results described in previous studies of the genes hrpb <dig> and hrpx corroborate the results we obtained for the mutants 02h <dig> and 03c <dig>  which carry mutations in the genes hrpb <dig> and hrpxct, respectively.
these two mutants caused no disease and their growth in citrus leaves was much lower than the xcc isolate  <dig> .
in xcv, hrpxv acts as a transcriptional activator for genes of the group hrp.
hrpxv is necessary for transcriptional activation of five hrp genes   <cit> .
the protein hrpb <dig> is necessary for the complete functionality of ttss, since hrpb <dig> mutants are not able to secrete avrbs <dig> or hrpb <dig> proteins in xcv  <cit> .
therefore, it can be assumed that these two mutants, 02h <dig> and 03c <dig>  lost their virulence because of their inability to take ttss factors to the host cell, which are necessary for growth in planta, since when these mutants are reactivated in culture media, cellular multiplication is similar to that of wild type.
another non-pathogenic mutant had mutated orf xac <dig>  which has similarity with the xyllela fastidiosa gene htra .
first identified in e. coli, the locus htra encodes a serine protease htra  that contains a catalytic triad  required for proteolytic activity and two pdz domains responsible for oligomerization of the protein complex, substrate recognition and substrate binding.
besides proteolytic activity, e. coli htra shows chaperone activity in vitro at low temperatures, where a conformational change of the protein masks the proteolytic residues.
at high temperatures, the catalytic residues are accessible and the proteolytic activity of htra prevails.
the htra proteases identified in e. coli are required for growth at 42°c and for the degradation of abnormally folded proteins in the periplasm.
it was later demonstrated that htra degrades heat-denatured proteins, in vivo and in vitro.
the very small amount of substrate for htra catalytic activity found in vivo suggests that the main biological role of the protein is the removal of nonnative, abnormally folded proteins from inside the cellular envelope.
in e. coli, htra is located in the periplasm, associated with the internal membrane  <cit> .
homologous htra proteins are found in most bacteria, and are well conserved throughout evolution.
their impact on bacterial physiology differs among the gram-negative bacteria.
in contrast to e. coli, htra is not essential for the growth of salmonella enterica serovar typhimurium at high temperatures, for instance.
the htra mutant of s. enterica serovar typhimurium showed reduced virulence in a murine model and reduced survival in macrophages.
the phenotypic characterization of htra s. enterica serovar typhimurium mutant revealed a decreased tolerance to oxidative stress, which can explain the reduced survival in macrophages, where reactive intermediates of oxygen are released during the oxidative explosion.
htra mutants of other gram-negative pathogenic bacteria, such as yersinia enterocolitica, klebsiella pneumoniae and brucella abortus, are sensitive to both high temperatures and oxidative stress  <cit> .
moreover, htra mutants of y. enterocolitica and of b. abortus show reduced virulence in murine models.
in listeria monocytogenes, transcriptional analyses in an htra mutant revealed that the gene htra is not induced in response to thermal shock, but rather to stress caused by low ph and penicillin g. in addition, a significant virulence decrease was detected in this mutant, revealing that htra is very important for the complete virulence of l. monocytogenes in mice.
recently, an htra mutant of l. monocytogenes 10403s was shown to be sensitive to oxidative stress and puromycin at high temperatures, and showed a reduced ability to produce biofilms and attenuated virulence in mice  <cit> .
however, the attenuated virulence of gram-negative htra mutants remains unclear since they are more susceptible to stress than the isolated parent is; the mutants may also be less viable in host tissues, which will trigger several types of stress to the invading cell.
besides, it is believed that the chaperone and processing functions of htra protein are necessary for folding secreted proteins, or that htra may be involved in the oligomerization and exportation of virulence factors  <cit> .
therefore, the htra gene has been shown to be essential for the complete virulence of many pathogens.
on the other hand, htra is not essential for bacterial growth under unstressed conditions, so it is a potential target for anti-pathogen drugs, including those that inhibit virulence rather than killing bacteria or stopping bacterial growth.
it is assumed that anti-pathogen drugs reduce the pressure for development of resistance, which is an extremely important trait when it comes to agricultural pests, because such a drug must be applied over large areas and produces high selection pressure.
moreover, not killing the target makes this kind of drug type ecologically sustainable, because it cannot favor bacterial evolution  <cit> .
thus, the data in the literature and the results of the present study allow the assumption, for the first time, that the gene htra  of xcc is part of an important process during pathogenic colonization, being necessary for the complete virulence of the pathogen.
heme groups are responsible for carrying out a wide variety of biological functions in prokaryotes and eukaryotes.
these groups are essential for respiration, oxygen metabolism and electron transport, as well as for prosthetic groups, hemoglobulins, hydroxylases, catalases, peroxidases and cytochromes  <cit> .
more recently, new roles for heme groups have been described as biosensors of diatomic gases  <cit>  and modulators of protein activity  <cit> .
protoheme biosynthesis involves seven enzymatic steps, starting from the universal precursor delta-aminolevulinic acid .
other heme groups that cells need are obtained from protoheme modifications.
in the step before production of protoheme, an iron ion  is inserted in protoporphyrin ix, catalyzed by ferrochelatase  <cit> .
one of the many roles played by protoheme in the cell is the constitution of cytochrome.
type c cytochromes, which contain a covalent heme c group, are widely distributed in organisms, in which they play a role in photosynthesis and electron transport from the respiratory chain.
most type c cytochromes of e. coli and s. enterica serovar typhimurium are c <dig> cytochromes, which are comprised of six covalently bound heme groups and are located in the periplasmic space where they act as dissimilatory nitrite reductase  <cit> .
therefore, heme groups are used in basic metabolism for energy production, in the electron transport chain in an aerobic pathway, and in the nitrite reduction complex in an anaerobic pathway.
the interruption of heme group production thus presumably affects the electron transport chain, which hinders the use of oxygen or nitrate as final electron recipients by cells.
if this hypothesis is true, it explains why the mutant 11d <dig>  which carries the interrupted orf xac <dig>  a delta-aminolevulinic dehydratase , does not cause disease and shows total absence of symptoms.
proteomic analysis showed that proteins involved in glycolytic and related pathways and fermentation are over-expressed in hemb mutant cells, which show exponential growth, compared to the parental strain  <cit> , indicating that the mutant hemb produces energy only from phosphorylation at the substrate level in vitro.
thus, the observation that the mutant 11d <dig> is multiplied in planta  is explained by the use of carbon sources for the production of anaerobic atp, or even by the use of the hemes produced by the plant.
so, considering the information available in the literature, the hemb mutant can survive in vitro and in planta by producing energy from hexoses or from intermediate compounds such as pyruvate, producing lactate, acetyl-coa, producing ethanol, or l-arginine, producing co <dig> + nh <dig>  moreover, its survival may also depend on the uptake of heme from the environment through specific carriers for this purpose present on xcc, ccma, ccmb, ccmc and cycz .
in the first three cases atp production is low and for the latter the possibility of spending a lot of energy to transport the heme compound into the cell, which also results in a low atp balance.
so, in all cases, the cell would probably optimize energy expenditure for survival.
whereas the process of pathogenesis demands the production of various enzymes, proteins and other compounds, these observations suggest that these processes will not be realized because of their high energy consumption.
consequently, although the cell survives reasonably well, both in vitro and in planta, it will not develop the disease and thus no external symptoms will be observed.
finally, the fact that the mutant hemb had a growth curve in planta very similar to wild type may be an indication that it is performing aerobic metabolism due to internalization of heme compounds from the host and not causing the disease because the energy balance is not favourable, since transport through the membrane consumes so much energy.
histidine kinases are proteins that can play a major process in bacterial metabolism.
these proteins, together with their cognate response regulators , can be part of two component systems , which constitute a signal transduction process in which bacteria sense, respond, and adapt to changes in their environment or intracellular state.
signal transduction starts when a histidine kinase senses a signal, e.g., by binding or reacting with a signaling molecule or due to a physical stimulus, and phosphorylates downstream proteins in the phosphorylation cascade that modulate the activity of a final set of protein targets, which then modulate protein activity or differential gene expression.
based on their components, two tcs exist: prototypical and phosphorelay systems  <cit> .
in the phosphorelay tcs pathway, a stimulus activates autophosphorylation of a hybrid histidine kinase, namely, a histidine kinase containing a phospho-accepting receiver domain, typically at the c-terminal end of the protein.
the catalytic and atpase  domain of the histidine kinase is responsible for binding atp and catalyzing autophosphorylation of a conserved histidine found within the dimerization and histidine phosphotransferase  domain.
the hiska domain mediates homodimerization and serves as the phosphodonor for a c-terminal receiver domain , similar to that found in response regulators.
a histidine phosphotransferase  then shuttles the phosphoryl group from the hybrid kinase to a soluble response regulator containing an output domain through protein-protein interaction or protein-dna interactions leading to differential gene expression  <cit> .
xcc has a gene that codes for a histidine kinase, xac <dig>  that is similar to the hybrid histidine kinase found in many tcs.
xac <dig> has hiska, hatpase, and response regulator domains .
an analysis using psort  <cit>  found that the predicted protein from xac <dig> is localized on the bacterial inner membrane and a blastp search result  <cit>  found that the first  <dig> amino acids only match sequences from x. citri subsp.
citri, x. campestris pv.
vesicatoria and x. oryzae pv.
oryzae, indicating that the n-terminal sequence is exclusive to xanthomonas.
the blastp result from amino acids  <dig> to  <dig> at the c-terminus found similarities with rpfc protein from xcc, and with many rpfc proteins that are involved in quorum sensing signaling mediated by a diffusible signal molecule dsf .
this quorum sensing mechanism plays a key role in the regulation of xanthan  biosynthesis, gene expression, motility, adaptation, and bacterial virulence  <cit> .
rpfc from xcc  has the same three domains: hiska, hatpase, and the response regulator, as well as an hpt domain.
furthermore, rpfc is a bacterial inner membrane protein  <cit> .
in xanthomonas, the rpfc and rpfg proteins are a two-component system implicated in dsf perception and signal transduction.
at a low cell density, the dsf sensor rpfc forms a complex with the dsf synthase rpff through its receiver domain, which prevents the enzyme from effective synthesis of the dsf signal.
in this step, dsf is synthesized at basal levels.
but when the cell density increases, extracellular dsf increases, too.
so at a high cell density, accumulated extracellular dsf interacts with rpfc and induces a conformational change in the sensor, which undergoes autophosphorylation and facilitates release of rpff and phosphorelay from the sensor to its response regulator rpfg.
now, rpff, together with rpfb, can induce the production of dsf, and rpfg can induce eps biosynthesis, gene expression, motility, adaptation, and bacterial virulence  <cit> .
the rpfc mutants produce significantly attenuated virulence factors, but synthesize about 16-fold higher dsf signal than the wild type  <cit> , whereas mutation of rpff or rpfb abolishes dsf production and results in reduced virulence factor production  <cit> .
deletion of either rpfc or rpfg decreases the production of eps and extracellular enzymes  <cit> .
based on these results, it was proposed that rpfc/rpfg is a signal transduction system that couples the synthesis of pathogenic factors to sensing of environmental signals that may include dsf itself  <cit> .
nevertheless, the current knowledge about the signal transduction pathway downstream of rpfc/rpfg is still little.
recent study presented evidence that the hd-gyp domain of rpfg is a cyclic di-gmp phosphodiesterase that degrades the second messenger bis--cyclic dimeric guanosine monophosphate  <cit> .
furthermore, rpfg interacts with ggdef domain-containing proteins  <cit> .
the same authors, andrade and coworkers, showed that rpfg interacts with sigma factor  <dig>  ntrbc, and other regulatory proteins.
we found a mutant, 18d <dig>  in our mutant library in which xac <dig> was knocked out; the mutation site is located inside the response regulator domain .
this mutant was observed at a high concentration in planta  but with no symptom development .
despite the ability of a hybrid histidine kinase to be involved in phosphorylation of any pathogenicity related gene, we believe that this protein plays a more sophisticated role in the virulence process in xcc.
considering the data presented above, namely a protein localized on the inner membrane with high similarity with rpfc, a xanthomonas exclusive amino terminus, and high mutant cells concentration in planta, led us to propose this role for xac <dig> in xcc: participation in the perception and transduction of signals in the quorum sensing system in this xanthomonas citri subsp.
citri.
besides these features, the fact that the response regulator domain  from xac <dig> interacts with the domains cheb_methylest , response_reg , trans_reg_c , ggdef , hpt , p <dig> , sigma54_activat , and antar   <cit>  gave us more data on which to base this hypothesis.
xac <dig> protein can be on the inner membrane and the amino terminus could act as a sensor to perceive host or environmental signals.
after signal reception, xac <dig> may be autophosphorylated.
the hiska domain serves as the phosphodonor for the c-terminal receiver domain .
a histidine phosphotransferase then shuttles the phosphoryl group from the hybrid kinase to a cytoplasmatic response regulator, which could be rpfg or another downstream protein in the signaling chain carrying at least one of the eight domains with which it could interact  <cit> .
thus, we are supposing that xac <dig> is an important required member of the signaling transduction process in xcc , acting together with rpfc/rpfg and required for complete virulence.
when rpfc, rpfg or xac <dig> is not functional, virulence is abolished, but the mutant is viable.
another observation that we think is important is the site of the mutation on xac3673: the response regulator domain.
the response regulator domain in rpfc and xac <dig> are very similar, indicating that they could share the same protein-protein interactions with rpfg or with other proteins in the downstream signaling pathway.
figure  <dig> summarizes our hypothesis about the proposed role of xac <dig> in quorum sensing in xcc.
finally, we compared the xcc genomic regions in which the mutated orfs are located to other bacterial genomes.
basically, we used the sequence analysis tool blast  <cit>  to compare these xcc regions with the corresponding regions of the genomes of five other xanthomonas species: x. campestris pv.
vesicatoria, x. oryzae pv.
oryzae maff, x. oryzae pv.
oryzae kacc <dig>  x. campestris pv.
campestris atcc  <dig> and x. campestris pv.
campestris  <dig>  at the end of this comparative analysis, five regions were highlighted .
region  <dig>  and region  <dig> , which hold respective knockout orfs xac <dig>  and xac <dig>  xac <dig> and xac <dig>  are exclusive to xcc.
however, regions  <dig>   <dig> and  <dig>  which contain respective knockout orfs xac <dig>  xac <dig> and xac <dig>  are present in at least one of the other studied genomes, but not in all .
in addition, some characteristics of these regions, such as abnormal variation in nucleotide composition  and the appearance of relaxases, mobilization proteins, phages, transposons and integrases , are good indicators of viable lateral transfer regions  <cit> .
indeed, recently lima and coworkers  <cit> , when examining the xcc genome in search of viable xcc genomic region candidates for lateral transfer regions, also concluded that regions  <dig> and  <dig>  are genomic islands, which supports the hypothesis.
the other three regions,  <dig>   <dig> and  <dig> , have no corresponding sequences or regions in the work of these authors, but regions  <dig> and  <dig> are very similar to the xauc <dig> and xauc <dig> regions identified by moreira and coworkers  <cit> .
being exclusive to xcc, regions  <dig> and  <dig> deserve special attention .
the xac <dig>  xac <dig> and xac <dig> orfs, which encode hypothetical proteins of unknown roles and that showed different expression patterns under the conditions mentioned above, are in region  <dig>  this region is found in the orf xac <dig>  and extends until xac <dig> , totaling  <dig>  kb.
in terms of composition, this region has mainly hypothetical orfs.
the encoded product of one of these orfs  interacts with the protein vird <dig>  a gene classically correlated with the type iv secretion system  <cit> .
it is important to emphasize that upstream of this region there are orfs that encode a virulence regulator  , transposases  and regulated component colsr .
most curious is the orf xac <dig>  which encodes an rhsd protein, and the respective mutants also show massive reduction of the necrosis phenotype , which also was upstream of region  <dig> .
in addition, for orfs xac <dig>  xac <dig> and xac <dig>  no classically described domain was found in the probable proteins encoded by these hypothetical orfs and an analysis by psort  <cit>  revealed that they are cytoplasmic proteins and, in a similar manner, no clusters of orthologous groups  of proteins  <cit>  were found, demonstrating that there is no similarity with any other sequences.
in a different way, region  <dig> also calls attention by containing  <dig> transposases, alternating with hypothetical orfs .
among orfs with functions previously predicted by genome annotations, there is orf xac <dig>  which encodes an fe-s oxidoreductase that has been knocked out, and another that encodes a hemolysin related protein .
for this orf, xac <dig>  it has also been proven experimentally that its product is connected to the vird <dig> product  <cit> .
related to the structural aspect, this region, besides having abnormal variations in the constitution of its nucleotides, is located between two major conserved gene clusters related to flagellum biosynthesis and regulation.
in other organisms, including some xanthomonas, these genes are concatenated, evidence that reinforces the hypothesis that this region was acquired by a lateral transfer process.
because of all of these peculiarities, these five regions qualify as strong candidates for classification as probable lateral transfer islands and, in this particular case, as probable pathogenicity islands, as they present many of the typical characteristics found in these regions  <cit> .
finally, another three orfs were analyzed under this profile : orf xac <dig>  which encodes a site-specific dna methyltransferase, is inserted in a region that is characterized as a phage insertion region; the sequence is partially found only in the two sequenced species of x. oryzae and in x. campestris atcc 33913; orf xac <dig>  which is in a region only found in x. vesicatoria; and orf xac <dig>  which encodes one transposase only absent in the x. vesicatoria strain.
in short, three of the seven orfs described as candidate genes to be present in lateral transfer islands were analyzed in terms of expression levels and conditions.
it was observed that they play important roles in plant-pathogen interrelations, because they are only expressed when cells are multiplied in planta.
the culture medium does not contain compounds present in plants, and for this reason, it did not induce expression.
however, the observation that mutants for these genes showed reduced virulence and symptom alterations supports their importance in the interaction with the host.
these results corroborate the altered pathogenicity of the mutants studied here when inoculated in a host plant, indicating that the products of these genes are important for pathogen establishment and development in the host.
CONCLUSIONS
the experiments described in the present study represent the first attempt to use a high-throughput mutagenesis analysis method to identify a wealth of genes that contribute to xcc virulence.
these results allowed identification of new putative virulence factors, as well as novel potential targets for drugs in this strain, especially the genes present in the xcc exclusive putative pathogenicity island.

 citrus canker is a disease caused by the phytopathogens xanthomonas citri subsp. citri, x. fuscans subsp. aurantifolli and x. alfalfae subsp. citrumelonis. among the three phytopathogens, the asiatic form, which causes citrus bacterial canker type a, is the most widely spread and severe, attacking all citrus varieties. in brazil, form a is the most important, being found in practically all areas where citrus canker has been detected. similarly to most phytobacterioses, there is no efficient way to control citrus canker. the only way to eliminate the disease is through the eradication of sick plants, a procedure that brings significant economical losses. by law, in são paulo state, the main citrus production area in brazil, it is mandated to eliminate all plants around the focus of infection in a m radius if the contaminated plants are less than % of the planting field and all plants in the planted field if the contaminated plants are more than %. in the latter case, cultivation is then prohibited in the area for the next years and there is no payment for lost production to the growers. considering the importance of the disease worldwide, especially for brazil, a brazilian group sequenced and annotated the complete genome of x. citri subsp. citri strain, which causes citrus canker, and compared it with x. campestris pv. campestris strain atcc the etiological agent of crucifer black rot. the citrus subspecies has open reading frames, of which % have been assigned function. in addition, xcc also has two plasmids that have genes, and for of them, no role has been proposed. although the genome of xcc has been characterized and annotated, the inferences made based on in silico analyses require experimental investigation to accurately detect which genes are related to the pathogen-host adaptation process, and which are associated with pathogenesis itself. therefore, functional genomics studies are necessary to elucidate the machinery required for pathogen installation and proliferation in plants, and the induction of citrus canker symptoms in the host. from the functional genomic perspective, large scale analysis of mutants by inoculation in host plants allows identification of the genes required for adaptation, pathogenesis and virulence, providing a best understanding of the colonization and infection potential of the bacteria. in this work, using transposon insertion mutagenesis, a library containing mutants of the citrus canker etiological agent x. citri subsp. citri strain was prepared and mutants were analyzed after individual inoculation of host plants. eight mutants with absent pathogenicity and mutants with reduced symptoms in planta, at varying intensities, were identified. mutated genes were identified by sequencing the total dna of the mutants with altered virulence, allowing the identification of the site of insertion of the transposon used for mutagenesis. a random selection of these genes was immobilized on a nylon membrane array and expression profiles were analyzed in vivo through nucleic acid hybridization to labeled cdna probes, using targets corresponding to wild xcc strains multiplied in non-infective or infective conditions. finally, a comparative genomic analysis of each mutated orf region from xcc with other sequenced xanthomonas genomes allowed the identification of five interesting genomic regions, with two being exclusive to xcc. the unique characteristics presented by these five regions suggest that they are probably new pathogenicity islands in xcc. the implications of the proteins encoded by these mutated orfs in host adaptation and colonization processes and citrus canker symptoms induction are discussed.  mutant analysis and in vivo pathogenicity test to identify and characterize genes involved in pathogenicity and virulence in x. citri subsp. citri isolate a library of mutants was built through random transposon insertion. to determine whether transposon insertion affected the ability of xcc to cause disease, mutants of this library were individually inoculated in rangpur lime plantlets. assuming the transposon is randomly distributed along the genome in a single-copy manner, the probability of finding one transposon insertion for a certain gene can be calculated by the formula: p = - n, where p is the probability of finding one transposon insert within a given gene; x is the length of the gene; g is the length of the genome; and n is the number of transposon inserts present in the population. based on the sequenced genome of citri and considering the main chromosome and two plasmids, the average length of each orf in the xcc genome is bp and the probability of finding one transposon insert for a certain gene is up to 47%. the mutants identified as having altered pathogenicity in this first round were re-inoculated and re-analyzed, resulting in a final mutants showing some symptomatic variation. the mutants were grouped in five classes according to severity of the major symptoms: total absence of symptoms; watersoaking; hyperplasia; necrosis; and hypersensitive-like response. the site of transposon insertion was determined by sequencing for all mutants. in mutants the transposon was inserted inside an orf and in four the insertion was at the 5'-end of the orf, probably in the promoter region. in addition, orfs were hit in two independent mutants and in two cases the same orf was hit in three different mutants, resulting in different orfs being hit. in all cases, mutants having a transposon insertion in the same orf, irrespective of the insertion site, showed the same phenotype as determined by independent evaluations at three different times. based on the classification proposed by the xcc genome group http://genoma fcav.unesp.br/xanthomonas, the mutated genes belong to several categories: seven participate in intermediary metabolism; three are classified in the biosynthesis of small molecules; three are involved in macromolecule metabolism; two are cell structure constituents; four participate in another cellular process; two are related to mobile genetic elements; four are involved with pathogenicity, virulence, and adaptation; eight are hypothetical orfs; and two are undefined orfs. therefore, among the mutants there are distinct mutated orfs. to verify that transposon insertion was random, one southern blot analysis was evaluated. it was possible to estimate that the number of xcc clones with double inserts in their genomes was approximately in every mutants of the library. the same analysis was done on all mutants and none of them had double inserts. analysis of the growth curve in planta and in vitro to analyze the behavior of some mutants in terms of growth in vitro and in planta, mutants were randomly selected and analyzed together with the wild type. although all mutants were inoculated with the same number of cells, including the wild-type strain, we observed cellular concentration differences after days of growth in citrus leaves. wild type showed cell growth until days, and from that point the growth curve in planta remained constant at close to cells/cm of leaf area. it was possible to group the mutants into five distinct patterns based on the numbers of cells per square cm: 1) mutants that showed a low concentration of cells during the infection period; 2) mutants that showed an average concentration of cells during the infection period; 3) mutants that had high concentrations of cells during the cellular infection period; 4) mutants that showed a sigmoid pattern of cell concentration around; and 5) mutants that had an increase in cell number equal to the wild type until the second day and then the concentration was stable until the 10th day, when it started to fall, reaching close to on the last day. furthermore, the mutant 18d also presented a sigmoid growth curve, but with a cell concentration above when the same mutants were grown in culture media, it was observed that the cells grew more similarly to the wild type over time. however, among all mutants tested, the 02h and 03c mutants, which in planta had lower cell concentrations, did not cause any symptoms. intriguingly, both genes are identified as involved with the type iii secretion system, reinforcing its importance in the disease induction process. gene expression to show that the mutated genes are related to the altered symptoms of disease and were involved in pathogenesis, the expression of orfs was analyzed through nucleic acid hybridization to labeled cdna probes in two situations: cells multiplied in culture medium and cells multiplied in citrus leaves. the results indicated that the orfs xac xac xac xac xac xac xac xac xac and xac are only expressed when xcc is multiplied in vivo; it was not possible to identify expression of these orfs when cells were multiplied in vitro. a single orf, xac showed no significant expression in any of the conditions. the two experimental replications showed similar results. discussion random mutagenesis through random transposon insertion in vivo in the genome has been widely and successfully used to study several microorganisms, whether pathogens or not. using this technique for pathogenicity and virulence studies of the causal agent of the citrus canker, a library with approximately viable mutants of x. citri subsp. citri isolate was obtained. through this strategy, the transposon/transposase complex was inserted directly into the cells through electroporation. southern blot analysis showed that % had a double transposon insertion, which is near that expected from the description accompanying the kit used to obtain mutants, where the rate of double inserts is about 1% of the clones. after individual inoculation of mutants in rangpur lime leaves, mutants were identified with some alteration in their ability to induce citrus canker symptoms. the mutated orfs in mutants with altered pathogenicity were identified through dna sequencing. in this group of mutants there were genes belonging to several functional categories, including genes previously known as being involved in the pathogenesis process, such as the proteins hrpb and uptc and new genes xac xac and xac the symptoms caused by these mutants were also widely variable, and eight of them did not cause disease, which was confirmed by the total absence of symptoms. in addition, to show that the mutated genes were really involved in the pathogenesis process, the expression of orfs was analyzed through nucleic acid hybridization using labeled cdna probes in two situations: cells multiplied in culture medium and cells multiplied in citrus leaves. the analyses of the blots showed that among these genes it was possible to observe the expression of most in planta, which denotes their importance in interaction or adaptation events during the infection process. however, no ptha mutant was identified, despite xcc having four distinct copies of ptha, two in each plasmid. it could be that mutation of just one ptha gene does not affect the establishment of xcc in either pathogenicity or symptoms. swarup and coworkers have shown that mutation in the ptha gene resulted in a complete loss of virulence on citrus, but the amino acid sequence coded by ptha is distinct from all four ptha copies present in xcc. we used homologous recombination to disrupt each copy of xcc ptha in order to determine the contribution of each copy to pathogenicity and virulence. however, this process is not trivial, because we would first have to obtain a null ptha mutant, ie, a mutant with all four copies of this gene mutated. under these conditions the adaptability of the null mutant could be tested, and, using that mutant, the contribution of each copy of ptha could be evaluated. another circumstance that may have influenced the absence of identified pthas mutants is the probability of having all the xcc genes mutated in our mutant library, which was only 47%, whereas empirically, it is much easier to hit the main chromosome, due to its size, than the plasmids. so, the probability of mutating a gene in the plasmid is also very small in relation to the probability of mutating a gene on the main chromosome. two of the non-virulent mutants carry genes previously described as being necessary for pathogenicity, hrpb and hrpxct; these two genes are part of the hrp system, which is present in most gram-negative phytopathogenic bacteria, except for agrobacterium, and is part of the ttss. many results indirectly suggest that virulence proteins, also called virulence effectors, are injected by the pathogen directly inside the host cells through a pilus. it is presumed that the effectual proteins stimulate or suppress several cellular functions of the host to benefit pathogen infection. in x. campestris pv. vesicatoria, the hrp cluster is kb and contains six operons, hrpa to hrpf. two regulator genes, hrpg and hrpx, located outside of the larger gene cluster, are responsible for activating the expression of hrp genes in planta and in xvm synthetic culture media. the mutant for hrpb in xcv was not able to cause disease in susceptible pepper plants or the hypersensitive reaction in pepper plants carrying the respective compatible r gene, in the presence of avr in the xcv isolate used in the study. subsequent studies confirmed that this protein, hrpb was not secreted; in other words, it is a protein that acts in the bacterial cell. in the same study, hrpb remained in the soluble protein extract while avrbs was secreted by the wild isolate, which was not observed in a hrpb defective mutant. the gene hrpxv was characterized and its function was determined. the amino acid sequence deduced indicated similarity with proteins of the arac family, which act in the regulation of gene expression. mutations at position of that gene stopped the resulting mutant from inducing disease symptoms in susceptible pepper and tomato plants and hr in resistant plants. complementation with fragments of that gene showed that only bp after the initiator codon is enough to produce a functional polypeptide. the cell concentration of hrpx mutants in planta revealed that the mutant had times less bacteria than the wild type genotype. these results described in previous studies of the genes hrpb and hrpx corroborate the results we obtained for the mutants 02h and 03c which carry mutations in the genes hrpb and hrpxct, respectively. these two mutants caused no disease and their growth in citrus leaves was much lower than the xcc isolate. in xcv, hrpxv acts as a transcriptional activator for genes of the group hrp. hrpxv is necessary for transcriptional activation of five hrp genes. the protein hrpb is necessary for the complete functionality of ttss, since hrpb mutants are not able to secrete avrbs or hrpb proteins in xcv. therefore, it can be assumed that these two mutants, 02h and 03c lost their virulence because of their inability to take ttss factors to the host cell, which are necessary for growth in planta, since when these mutants are reactivated in culture media, cellular multiplication is similar to that of wild type. another non-pathogenic mutant had mutated orf xac which has similarity with the xyllela fastidiosa gene htra. first identified in e. coli, the locus htra encodes a serine protease htra that contains a catalytic triad required for proteolytic activity and two pdz domains responsible for oligomerization of the protein complex, substrate recognition and substrate binding. besides proteolytic activity, e. coli htra shows chaperone activity in vitro at low temperatures, where a conformational change of the protein masks the proteolytic residues. at high temperatures, the catalytic residues are accessible and the proteolytic activity of htra prevails. the htra proteases identified in e. coli are required for growth at 42°c and for the degradation of abnormally folded proteins in the periplasm. it was later demonstrated that htra degrades heat-denatured proteins, in vivo and in vitro. the very small amount of substrate for htra catalytic activity found in vivo suggests that the main biological role of the protein is the removal of nonnative, abnormally folded proteins from inside the cellular envelope. in e. coli, htra is located in the periplasm, associated with the internal membrane. homologous htra proteins are found in most bacteria, and are well conserved throughout evolution. their impact on bacterial physiology differs among the gram-negative bacteria. in contrast to e. coli, htra is not essential for the growth of salmonella enterica serovar typhimurium at high temperatures, for instance. the htra mutant of s. enterica serovar typhimurium showed reduced virulence in a murine model and reduced survival in macrophages. the phenotypic characterization of htra s. enterica serovar typhimurium mutant revealed a decreased tolerance to oxidative stress, which can explain the reduced survival in macrophages, where reactive intermediates of oxygen are released during the oxidative explosion. htra mutants of other gram-negative pathogenic bacteria, such as yersinia enterocolitica, klebsiella pneumoniae and brucella abortus, are sensitive to both high temperatures and oxidative stress. moreover, htra mutants of y. enterocolitica and of b. abortus show reduced virulence in murine models. in listeria monocytogenes, transcriptional analyses in an htra mutant revealed that the gene htra is not induced in response to thermal shock, but rather to stress caused by low ph and penicillin g. in addition, a significant virulence decrease was detected in this mutant, revealing that htra is very important for the complete virulence of l. monocytogenes in mice. recently, an htra mutant of l. monocytogenes 10403s was shown to be sensitive to oxidative stress and puromycin at high temperatures, and showed a reduced ability to produce biofilms and attenuated virulence in mice. however, the attenuated virulence of gram-negative htra mutants remains unclear since they are more susceptible to stress than the isolated parent is; the mutants may also be less viable in host tissues, which will trigger several types of stress to the invading cell. besides, it is believed that the chaperone and processing functions of htra protein are necessary for folding secreted proteins, or that htra may be involved in the oligomerization and exportation of virulence factors. therefore, the htra gene has been shown to be essential for the complete virulence of many pathogens. on the other hand, htra is not essential for bacterial growth under unstressed conditions, so it is a potential target for anti-pathogen drugs, including those that inhibit virulence rather than killing bacteria or stopping bacterial growth. it is assumed that anti-pathogen drugs reduce the pressure for development of resistance, which is an extremely important trait when it comes to agricultural pests, because such a drug must be applied over large areas and produces high selection pressure. moreover, not killing the target makes this kind of drug type ecologically sustainable, because it cannot favor bacterial evolution. thus, the data in the literature and the results of the present study allow the assumption, for the first time, that the gene htra of xcc is part of an important process during pathogenic colonization, being necessary for the complete virulence of the pathogen. heme groups are responsible for carrying out a wide variety of biological functions in prokaryotes and eukaryotes. these groups are essential for respiration, oxygen metabolism and electron transport, as well as for prosthetic groups, hemoglobulins, hydroxylases, catalases, peroxidases and cytochromes. more recently, new roles for heme groups have been described as biosensors of diatomic gases and modulators of protein activity. protoheme biosynthesis involves seven enzymatic steps, starting from the universal precursor delta-aminolevulinic acid. other heme groups that cells need are obtained from protoheme modifications. in the step before production of protoheme, an iron ion is inserted in protoporphyrin ix, catalyzed by ferrochelatase. one of the many roles played by protoheme in the cell is the constitution of cytochrome. type c cytochromes, which contain a covalent heme c group, are widely distributed in organisms, in which they play a role in photosynthesis and electron transport from the respiratory chain. most type c cytochromes of e. coli and s. enterica serovar typhimurium are c cytochromes, which are comprised of six covalently bound heme groups and are located in the periplasmic space where they act as dissimilatory nitrite reductase. therefore, heme groups are used in basic metabolism for energy production, in the electron transport chain in an aerobic pathway, and in the nitrite reduction complex in an anaerobic pathway. the interruption of heme group production thus presumably affects the electron transport chain, which hinders the use of oxygen or nitrate as final electron recipients by cells. if this hypothesis is true, it explains why the mutant 11d which carries the interrupted orf xac a delta-aminolevulinic dehydratase, does not cause disease and shows total absence of symptoms. proteomic analysis showed that proteins involved in glycolytic and related pathways and fermentation are over-expressed in hemb mutant cells, which show exponential growth, compared to the parental strain, indicating that the mutant hemb produces energy only from phosphorylation at the substrate level in vitro. thus, the observation that the mutant 11d is multiplied in planta is explained by the use of carbon sources for the production of anaerobic atp, or even by the use of the hemes produced by the plant. so, considering the information available in the literature, the hemb mutant can survive in vitro and in planta by producing energy from hexoses or from intermediate compounds such as pyruvate, producing lactate, acetyl-coa, producing ethanol, or l-arginine, producing co + nh moreover, its survival may also depend on the uptake of heme from the environment through specific carriers for this purpose present on xcc, ccma, ccmb, ccmc and cycz. in the first three cases atp production is low and for the latter the possibility of spending a lot of energy to transport the heme compound into the cell, which also results in a low atp balance. so, in all cases, the cell would probably optimize energy expenditure for survival. whereas the process of pathogenesis demands the production of various enzymes, proteins and other compounds, these observations suggest that these processes will not be realized because of their high energy consumption. consequently, although the cell survives reasonably well, both in vitro and in planta, it will not develop the disease and thus no external symptoms will be observed. finally, the fact that the mutant hemb had a growth curve in planta very similar to wild type may be an indication that it is performing aerobic metabolism due to internalization of heme compounds from the host and not causing the disease because the energy balance is not favourable, since transport through the membrane consumes so much energy. histidine kinases are proteins that can play a major process in bacterial metabolism. these proteins, together with their cognate response regulators, can be part of two component systems, which constitute a signal transduction process in which bacteria sense, respond, and adapt to changes in their environment or intracellular state. signal transduction starts when a histidine kinase senses a signal, e.g., by binding or reacting with a signaling molecule or due to a physical stimulus, and phosphorylates downstream proteins in the phosphorylation cascade that modulate the activity of a final set of protein targets, which then modulate protein activity or differential gene expression. based on their components, two tcs exist: prototypical and phosphorelay systems. in the phosphorelay tcs pathway, a stimulus activates autophosphorylation of a hybrid histidine kinase, namely, a histidine kinase containing a phospho-accepting receiver domain, typically at the c-terminal end of the protein. the catalytic and atpase domain of the histidine kinase is responsible for binding atp and catalyzing autophosphorylation of a conserved histidine found within the dimerization and histidine phosphotransferase domain. the hiska domain mediates homodimerization and serves as the phosphodonor for a c-terminal receiver domain, similar to that found in response regulators. a histidine phosphotransferase then shuttles the phosphoryl group from the hybrid kinase to a soluble response regulator containing an output domain through protein-protein interaction or protein-dna interactions leading to differential gene expression. xcc has a gene that codes for a histidine kinase, xac that is similar to the hybrid histidine kinase found in many tcs. xac has hiska, hatpase, and response regulator domains. an analysis using psort found that the predicted protein from xac is localized on the bacterial inner membrane and a blastp search result found that the first amino acids only match sequences from x. citri subsp. citri, x. campestris pv. vesicatoria and x. oryzae pv. oryzae, indicating that the n-terminal sequence is exclusive to xanthomonas. the blastp result from amino acids to at the c-terminus found similarities with rpfc protein from xcc, and with many rpfc proteins that are involved in quorum sensing signaling mediated by a diffusible signal molecule dsf. this quorum sensing mechanism plays a key role in the regulation of xanthan biosynthesis, gene expression, motility, adaptation, and bacterial virulence. rpfc from xcc has the same three domains: hiska, hatpase, and the response regulator, as well as an hpt domain. furthermore, rpfc is a bacterial inner membrane protein. in xanthomonas, the rpfc and rpfg proteins are a two-component system implicated in dsf perception and signal transduction. at a low cell density, the dsf sensor rpfc forms a complex with the dsf synthase rpff through its receiver domain, which prevents the enzyme from effective synthesis of the dsf signal. in this step, dsf is synthesized at basal levels. but when the cell density increases, extracellular dsf increases, too. so at a high cell density, accumulated extracellular dsf interacts with rpfc and induces a conformational change in the sensor, which undergoes autophosphorylation and facilitates release of rpff and phosphorelay from the sensor to its response regulator rpfg. now, rpff, together with rpfb, can induce the production of dsf, and rpfg can induce eps biosynthesis, gene expression, motility, adaptation, and bacterial virulence. the rpfc mutants produce significantly attenuated virulence factors, but synthesize about 16-fold higher dsf signal than the wild type, whereas mutation of rpff or rpfb abolishes dsf production and results in reduced virulence factor production. deletion of either rpfc or rpfg decreases the production of eps and extracellular enzymes. based on these results, it was proposed that rpfc/rpfg is a signal transduction system that couples the synthesis of pathogenic factors to sensing of environmental signals that may include dsf itself. nevertheless, the current knowledge about the signal transduction pathway downstream of rpfc/rpfg is still little. recent study presented evidence that the hd-gyp domain of rpfg is a cyclic di-gmp phosphodiesterase that degrades the second messenger bis--cyclic dimeric guanosine monophosphate. furthermore, rpfg interacts with ggdef domain-containing proteins. the same authors, andrade and coworkers, showed that rpfg interacts with sigma factor ntrbc, and other regulatory proteins. we found a mutant, 18d in our mutant library in which xac was knocked out; the mutation site is located inside the response regulator domain. this mutant was observed at a high concentration in planta but with no symptom development. despite the ability of a hybrid histidine kinase to be involved in phosphorylation of any pathogenicity related gene, we believe that this protein plays a more sophisticated role in the virulence process in xcc. considering the data presented above, namely a protein localized on the inner membrane with high similarity with rpfc, a xanthomonas exclusive amino terminus, and high mutant cells concentration in planta, led us to propose this role for xac in xcc: participation in the perception and transduction of signals in the quorum sensing system in this xanthomonas citri subsp. citri. besides these features, the fact that the response regulator domain from xac interacts with the domains cheb_methylest, response_reg, trans_reg_c, ggdef, hpt, p, sigma54_activat, and antar gave us more data on which to base this hypothesis. xac protein can be on the inner membrane and the amino terminus could act as a sensor to perceive host or environmental signals. after signal reception, xac may be autophosphorylated. the hiska domain serves as the phosphodonor for the c-terminal receiver domain. a histidine phosphotransferase then shuttles the phosphoryl group from the hybrid kinase to a cytoplasmatic response regulator, which could be rpfg or another downstream protein in the signaling chain carrying at least one of the eight domains with which it could interact. thus, we are supposing that xac is an important required member of the signaling transduction process in xcc, acting together with rpfc/rpfg and required for complete virulence. when rpfc, rpfg or xac is not functional, virulence is abolished, but the mutant is viable. another observation that we think is important is the site of the mutation on xac3673: the response regulator domain. the response regulator domain in rpfc and xac are very similar, indicating that they could share the same protein-protein interactions with rpfg or with other proteins in the downstream signaling pathway. figure summarizes our hypothesis about the proposed role of xac in quorum sensing in xcc. finally, we compared the xcc genomic regions in which the mutated orfs are located to other bacterial genomes. basically, we used the sequence analysis tool blast to compare these xcc regions with the corresponding regions of the genomes of five other xanthomonas species: x. campestris pv. vesicatoria, x. oryzae pv. oryzae maff, x. oryzae pv. oryzae kacc x. campestris pv. campestris atcc and x. campestris pv. campestris at the end of this comparative analysis, five regions were highlighted. region and region, which hold respective knockout orfs xac and xac xac and xac are exclusive to xcc. however, regions and which contain respective knockout orfs xac xac and xac are present in at least one of the other studied genomes, but not in all. in addition, some characteristics of these regions, such as abnormal variation in nucleotide composition and the appearance of relaxases, mobilization proteins, phages, transposons and integrases, are good indicators of viable lateral transfer regions. indeed, recently lima and coworkers, when examining the xcc genome in search of viable xcc genomic region candidates for lateral transfer regions, also concluded that regions and are genomic islands, which supports the hypothesis. the other three regions, and, have no corresponding sequences or regions in the work of these authors, but regions and are very similar to the xauc and xauc regions identified by moreira and coworkers. being exclusive to xcc, regions and deserve special attention. the xac xac and xac orfs, which encode hypothetical proteins of unknown roles and that showed different expression patterns under the conditions mentioned above, are in region this region is found in the orf xac and extends until xac, totaling kb. in terms of composition, this region has mainly hypothetical orfs. the encoded product of one of these orfs interacts with the protein vird a gene classically correlated with the type iv secretion system. it is important to emphasize that upstream of this region there are orfs that encode a virulence regulator, transposases and regulated component colsr. most curious is the orf xac which encodes an rhsd protein, and the respective mutants also show massive reduction of the necrosis phenotype, which also was upstream of region. in addition, for orfs xac xac and xac no classically described domain was found in the probable proteins encoded by these hypothetical orfs and an analysis by psort revealed that they are cytoplasmic proteins and, in a similar manner, no clusters of orthologous groups of proteins were found, demonstrating that there is no similarity with any other sequences. in a different way, region also calls attention by containing transposases, alternating with hypothetical orfs. among orfs with functions previously predicted by genome annotations, there is orf xac which encodes an fe-s oxidoreductase that has been knocked out, and another that encodes a hemolysin related protein. for this orf, xac it has also been proven experimentally that its product is connected to the vird product. related to the structural aspect, this region, besides having abnormal variations in the constitution of its nucleotides, is located between two major conserved gene clusters related to flagellum biosynthesis and regulation. in other organisms, including some xanthomonas, these genes are concatenated, evidence that reinforces the hypothesis that this region was acquired by a lateral transfer process. because of all of these peculiarities, these five regions qualify as strong candidates for classification as probable lateral transfer islands and, in this particular case, as probable pathogenicity islands, as they present many of the typical characteristics found in these regions. finally, another three orfs were analyzed under this profile: orf xac which encodes a site-specific dna methyltransferase, is inserted in a region that is characterized as a phage insertion region; the sequence is partially found only in the two sequenced species of x. oryzae and in x. campestris atcc 33913; orf xac which is in a region only found in x. vesicatoria; and orf xac which encodes one transposase only absent in the x. vesicatoria strain. in short, three of the seven orfs described as candidate genes to be present in lateral transfer islands were analyzed in terms of expression levels and conditions. it was observed that they play important roles in plant-pathogen interrelations, because they are only expressed when cells are multiplied in planta. the culture medium does not contain compounds present in plants, and for this reason, it did not induce expression. however, the observation that mutants for these genes showed reduced virulence and symptom alterations supports their importance in the interaction with the host. these results corroborate the altered pathogenicity of the mutants studied here when inoculated in a host plant, indicating that the products of these genes are important for pathogen establishment and development in the host.  the experiments described in the present study represent the first attempt to use a high-throughput mutagenesis analysis method to identify a wealth of genes that contribute to xcc virulence. these results allowed identification of new putative virulence factors, as well as novel potential targets for drugs in this strain, especially the genes present in the xcc exclusive putative pathogenicity island. 
finally, comparative genomic analysis revealed that mutated orfs are in new putative pathogenicity islands. nucleic acid hybridization using labeled cdna probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis. citrus canker is a disease caused by the phytopathogens xanthomonas citri subsp. the first of the three species, which causes citrus bacterial canker type a, is the most widely spread and severe, attacking all citrus species. like most phytobacterioses, there is no efficient way to control citrus canker. among the mutants with no canker symptoms are the hrpb and hrpx genes, two genes that belong to type iii secretion system, two hypothetical orfs and, surprisingly, the htra gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. their ability to cause citrus canker was analyzed every days until days after inoculation; a set of mutants showed altered virulence, with presenting a complete loss of causing citrus canker symptoms. in brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. the identification of these new genes related with xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker. sequencing of the insertion site in all mutants revealed that different orfs were hit, since some orfs were hit in more than one mutant, with mutants for the same orf presenting the same phenotype. an analysis of these orfs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with xanthomonas pathogenicity before, including hypothetical orfs. through transposon insertion mutagenesis, mutants of xanthomonas citri subsp.
 citrus canker is a disease caused by the phytopathogens xanthomonas citri subsp. citri, xanthomonas fuscans subsp. aurantifolli and xanthomonas alfalfae subsp. citrumelonis. the first of the three species, which causes citrus bacterial canker type a, is the most widely spread and severe, attacking all citrus species. in brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. like most phytobacterioses, there is no efficient way to control citrus canker. considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.  through transposon insertion mutagenesis, mutants of xanthomonas citri subsp. citri strain were obtained, and were inoculated in rangpur lime leaves. their ability to cause citrus canker was analyzed every days until days after inoculation; a set of mutants showed altered virulence, with presenting a complete loss of causing citrus canker symptoms. sequencing of the insertion site in all mutants revealed that different orfs were hit, since some orfs were hit in more than one mutant, with mutants for the same orf presenting the same phenotype. an analysis of these orfs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with xanthomonas pathogenicity before, including hypothetical orfs. among the mutants with no canker symptoms are the hrpb and hrpx genes, two genes that belong to type iii secretion system, two hypothetical orfs and, surprisingly, the htra gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. nucleic acid hybridization using labeled cdna probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. finally, comparative genomic analysis revealed that mutated orfs are in new putative pathogenicity islands.  the identification of these new genes related with xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker. 