Citrus canker is caused by various species of the bacterial genus Xanthomonas, and the most aggressive canker is induced by Xanthomonas citri subsp. citri pathotype A (Xac = Xanthomonas axonopodis pv. citri)  , which is responsible for the so-called Asiatic or type A Canker. The disease symptoms evolve as initial water soaking followed by hyperplasia and necrosis at the site of infection, lead to the dispersion of the pathogen to other infection sites across plant host tissues  . Given its importance as a pan-tropical plague, Xac genome was completely sequenced  . Since then, conducting large-scale studies has prioritized the functional elucidation of genes associated with adaptation to host citrus plants, or those involved in virulence and/or pathogenesis.
Although less effective than Xac, it is worth mentioning that the variants X. citri subsp. citri A* and AW, as well as X. fuscans subsp. aurantifolli strains B and C also cause citrus canker    . Their importance is attributed to their ability to infect only specific species of citrus, since they are restricted to certain regions in the world or vary considerably in their virulence induction profiles which are generally weaker than that observed for canker A. For these reasons, the genomes of these bacteria were also completely or partially sequenced    , thus enabling a comparative genomic analysis would guide the search for genes that possibly explain the role of biological variants, leading to a better characterization of the pathosystem as well as its relationships with respective hosts.
With the aim of gaining a better understanding of the repertoire of genes and proteins associated with Xac adaptation and virulence-induction in citrus, a series of studies have described the effect of mutations in specific genes impairing pathogen virulence and physiological responses of plant host under pathogen infection      . However, there are few published studies that jointly describe the discovery of a new gene repertoire related to pathogenicity and virulence in the Xac genome, which represents a breakthrough in the understanding of the relationship of this phytopathogen with its compatible hosts. A previous study based on the in planta analysis of Xac pathotype A mutants has led to the identification of 44 mutated genes in strains that show total or partial inability to induce the Xac virulence phenotype in the host plant  . Another study involving the same organism described 292 mutants showing a massive reduction in virulence phenotypes  . More recently, seven new genes associated with virulence in X. citri subsp. citri 29-1 have been also isolated from a library of mutants  .
As the random mutant in planta screening approach has been proved to be useful for the discovery of novel genes involved in Xac pathogenicity and virulence processes, in the present study, a new set of 1300 random Xac mutant strains are inoculated into citrus host plants, and twenty-six strains have showed none or reduced canker symptoms induced in infected hosts, being the mutated loci to these mutants identified by sequencing. A quantitative assessment comparing wild type Xac with mutants through in planta growth curves pointed to remarkable reductions on the proliferation of mutant strains inside host infected tissue. Beside functionally assigned mutated genes, special emphasis was given to eleven hypothetical genes, never before related to adaptation or virulence processes in this patho system.
2. Materials & Methods
2.1. Bacterial Strains and Growth Conditions
The wild type Xac strain 306 (wt), obtained from the phytopathogenic bacteria collection of the Laboratory of Bacteriology of the Agronomical Institute (IAPAR, Paraná state, Brazil), was stored in agar slant tube containing phosphate buffered medium (calcium nitrate 0.5 g/L, ferrous sulfate 0.5 g/L, sodium phosphate dibasic 2 g/L, peptone 5 g/L, sucrose 20 g/L, agar 20 g/L and adjusted to pH 6.6) at room temperature. Mutants obtained by random insertion using the EZ::Tn5 < KAN-2 > Tnp Transposome Kit (Epicentre Technologies), were maintained in Glycerol-TS growing medium (tryptone 10 g/L, sucrose 10 g/L, sodium glutamate 1 g/L, glycerol 150 ml/L and kanamycin antibiotic 100 μg/mL) at −80˚C. The growth experiments for Xac wt and mutants were performed in TSA medium (tryptone 10 g/L, sucrose 10 g/L and monosodium glutamate 1 g/L) at 28˚C, with addition of agar (15 g/L) where solid medium was required. Cells were grown over night in test tubes containing 3 mL of culture medium, at 28˚C with shaking at 180 rpm before inoculation. In the case of mutants, kanamycin was added to the culture medium to final concentration of 100 μg/mL for solid medium or 50 μg/mL for liquid medium.
2.2. In Vitro Mutagenesis
A set of Xac strain 306 mutants was obtained by random insertion of the Tn5 transposon   with an EZ::Tn5 KAN-2 Tnp Transposome Kit™, according to the instructions of the manufacturer (Epicentre Technologies). The transposon was inserted by electroporation (2500 V, 25 μF, 200 ohms, 0.2 cm cuvette width). Transformed colonies were selected on solid TS culture medium containing kanamycin and mutants were picked and transferred individually to 96-well microtitre plates containing liquid TS culture medium with kanamycin and 20% (v/v) glycerol. After growing for 2 days at 28˚C with shaking at 200 rpm, the plates were stored at −80˚C.
2.3. In Vivo Virulence Test
The mutants were individually multiplied in solid TS medium with kanamycin, while the wild-type strain (wt) was multiplied in medium without kanamycin. After growth at 28˚C for 48 h, the mutants and wt strain were individually resuspended in 1 mM MgCl2 end each had their cellular concentration adjusted to an optical density of 0.3 at 600 nm, corresponding to approximately 108 CFU/mL  . The bacterial suspension was infiltrated by pressing with a needleless syringe in one point of the left abaxial side of young Citrus sinensis end Citrus limonia leaves. The wt strain (control) was inoculated on the right side of the same leaf using the same concentration and conditions. After inoculation, plants were grown in a room at 28˚C with artificial light 3000 lux with 12 h photoperiod by 16 days. Evaluation of symptoms was carried out over 16 days (4, 8, 12 and 16 days after inoculation) and documented by digital photography. Those mutants that were unable to induce the pathogenic process or led to a difference in the symptoms, compared to that observed for the wt strain were selected.
2.4. Identification of Mutated Genes
The identification of mutated genes follows all procedures and steps (DNA extraction, transformation of E. coli with the recombinant plasmid, sequencing of mutated genes and Southern blotting validation) previously described by  .
2.5. Bacterial in Planta Growth Curves
In planta growth curve experiments were carried out with Xac wt and nine mutants with altered virulence (XAC0068, XAC0732, XAC2265, XAC3278, XAC0075, XAC1491, XAC∆aslB, XAC2457 and XAC3906). Three different clones (biological replicates) of each mutant or wt strains were multiplied in solid TS culture medium as described above, with the addition of kanamycin for mutant clones. After growth at 28˚C for 48 h, each culture was individually resuspended in 1 mM MgCl2 and the cellular concentration was adjusted to an optical density of 0.001 at 600 nm (105 CFU/mL). Then, each triplicate of bacterial suspension for a given strain (mutants or wt) was infiltrated at three distinct locations (technical replicates) on the abaxial side of young Citrus limonia leaves using a 1cc needleless tuberculin syringe and the inoculated area per leaf was enough to take a disk of 0.75 cm in diameter. Quantitative analyses were performed 0, 2, 4, 6, 8, 10, 12 and 14 days after inoculation. At each time, 3 disks of 0.75 cm in diameter were removed from different inoculated leaves for each clone (three biological replicates) and transferred to an eppendorf tube containing 1 mL of 1 mM MgCl2 cold solution. Immediately, each leaf disk was ground individually on ice with a pestle. Serial dilutions of 10−1 to 10−7 were prepared and 10 μL of each dilution was dropped on three plates (technical triplicate) of solid TS medium containing kanamycin (except for the wt strain) using the microculture technique. Plates were kept at 28˚C for 36 hours, and isolated colonies were counted. The results were expressed as the average of the three biological replicates (with three technical triplicate) ± standard deviation. The presented results correspond to the arithmetic average of nine repetitions for each analyzed strain.
2.6. Analysis of Orthologous Genes and Evolutive Inference of Disrupted Genes
The nucleotide sequence of twenty-six Xac disrupted gene were compared to non-re- dundant database (nr) using the blastx alghoritm  to search for the presence of orthologous of these genes in twenty-five genomes belonging to four genera of Xanthomonadaceae family: Xanthomonas, Xylella, Stenotrophomonas and Pseudoxanthomonas, with all the genomes been present in the Kegg database  . An E-value lower than 1e−20, was the criteria to consider a gene presenting in the chosen genomes (Table 1).
A presence-absence matrix was constructed and analyzed using the Tree View software (Page, 1996), selecting the heat map option. To the heat map result was incorporated an analysis based on the production of two dendograms based on presence and absence of genes by species and based on the distribution of orthologous genes in a given specie. This allowed defining 4 sets of orthologous genes in the genomes. The evolutionary analysis of these orthologous genes was done by parsimony analysis, using a vertical cladogram where selected strains represent each one of the twenty-five ge-
Table 1. General information of genomes used in this study.
nomes, already grouped according to the profile of taxa separation based on 16S-rGNA or gyrB gene sequences   , and the analysis were based on the presence or absence of the 26 mutated genes in the genomes.
3. Results and Discussion
Studies carried out with random mutant libraries proved to be important tools for the discovery of new gene functions or the involvement of genes with specific functions, already defined in other essential biological processes, for the survival of pathogens within the plant, or virulence induction. The in planta screening of 1300 Xac mutant strains, allowed the selection of twenty-six on the basis of defective canker symptoms induced in infected hosts and evaluated in terms of the structure and composition of their respective mutated genes, in addition to their location and organization in the Xac genome. These results brought improvement to our understanding about the origin of these genes, especially with respect to genes categorized as hypothetical, which corresponded to 42.3% (n = 11) of these mutants (Figure 1(a)). The analysis of the genomic region where these genes are located revealed that three genes defined as hypothetical are located into putative lateral gene transfer islands (Figure 1(b)). The remaining 15 knockout genes were categorized as showing putative functions. Importance was given to genes related to DNA replication or repair, which accounted for a total of 4 mutants
Figure 1. Mutants general features after inoculation in Citrus sinensis and Citrus limonia hosts. (a) Classification of mutants into phenotypical categories: 1―Nonpathogenic mutants; 2―Mu- tants showing partial loss of pathogenicity; 3―Mutants with partial loss of necrosis and total loss of hyperplasia; 4―Mutants presenting decreased hyperplasia and/or necrosis; 5―Mutants showing increased necrosis. (b) Localization of the disrupted genes in XAC genome. The likely genomic islands (red strips) were determined by the program IslandViewer (Dhillon, et al. 2013). Each one of the twenty-six mutants is pointed by triangles on the circle representing XAC chromosome. ((a) and (b)) HG―Hypothetical gene (green); WFA―With function assigned (red).
(15.4%). Based on the analysis of the composition of protein domains associated with genes defined as hypothetical, it was also possible to understand and assign potential functions to their encoded proteins. For example, although the protein product of the gene XAC4321 has low complexity, it exhibits another domain that encodes a signal peptide, thus suggesting its potential to be secreted by the bacterium (Table 2).
To facilitate the functional and structural description, the selected mutants were classified into five categories based on observed changes in their phenotypic profile (total or partial absence of the virulence phenotype) and infected host (C. sinensis and C. limonia).
3.1. Mutants Showing Total Loss of Virulence in C. sinensis and C. limonia
Out of twenty-six mutants analyzed, four (15.4%) knockouts led to a complete loss of virulence in both evaluated hosts (Table 2, Figure 2), three of them correspond to hypothetical genes (∆XAC0068, ∆XAC0732 and ∆XAC3278), and one is predicted to encode a helicase IV (XAC∆helD).
In plant growth curves analysis conducted to the strain ∆XAC0068, corresponding to a mutated hypothetical gene, it has shown less than 1000 times smaller growth in the citrus leaf parenchyma when compared to Xac wild type strain. This clearly demonstrates that the mutation affected the adaptive process of this strain within the inner intercellular spaces of the host plant (Figure 2(a)). Sequence analysis of the XAC0068 gene using blastx  demonstrated 99% to 76% identity with four other genes present in different bacteria: XAC29_00345, XCAW_00457, Acav_1091, F11_07645 and Rru_A1480. The first two genes correspond to orthologs of X. axonopodis Xac29-1  and X. citri subsp. citri Aw12879  , both canker-causing agents in citrus plants. The third organism with an orthologous sequence was Acidovorax avenae subsp. avenae, a member of the beta-proteobacteria responsible for causing disease in many plants of economic importance, such as rice, oats, sugar cane, and corn  . The fourth organism in which an ortholog was identified was Rhodospirillum rubrum, an autotrophic alpha-proteobacterium which inhabits a wide variety of environments such as lakes, streams, mud and sewage   . The common feature among these four orthologs is the presence of the NTP_transf_5 motif, belonging to the nucleotidyl transferase group involved in DNA repair  . It is interesting to notice that these similarities found in three different classes of bacteria (gamma, beta and alpha) belonging to such diverse ecological niches could suggest the occurrence of horizontal gene transfer between these species. This hypothesis is supported by the fact that no orthologs of XAC0068 gene was found in any other species of the genus Xanthomonas other than those mentioned above, ruling out the possibility of vertical inheritance. The presence of this gene in the genomes of species related to canker disease might indicate a functional role during interaction with citrus hosts.
Growth curve analysis for ∆XAC0732 mutant compared to wild type profile has shown a 102 reduction in bacterial titration, thus suggesting virulence impairment (Figure 2(b)). The gene knocked out in this mutant proved to be unique to the genus Xanthomonas, since blastx analysis  showed similarity with only five hypothetical
Table 2.General characteristics of studied mutants.
Figure 2. Phenotype and growth curve in vivo of the mutants that led to total loss of the virulence in Citrus sinensis and Citrus limonia. In each of the four analyzed mutants ((a) to (d)), it’s possible to follow the virulence progression over the 16 days in C. sinensis (at days 4, 8, 12 and 16 after infection), while for C. limonia only the virulence phenotype at the 16 days after infection is presented. The in vivo growth curve presented is for C. sinensis.
genes of this genus: XAC29_03725, XCAW_03850, XFF4834R_chr07400, XACM_0730 and XCV0786. The two sequences with higher degrees of identity (99%) were again found in X. axonopodis Xac29-1 and X. citri subsp. citri Aw12879, respectively  , which are strains capable of inducing canker symptoms in citrus hosts. The third ortholog was found in X. fuscans subsp. fuscans, a specie capable of causing damage in leguminous plants, among which bean as the most important one  . The fourth orthologous sequence was found in X. alfalfae subsp. citrumelonis (X. axonopodis pv. citrumelo), which specifically attacks rootstocks of the ‘Swingle’ citrumelo (Citrus paradisi x Poncirus trifoliata)  , and the fifth ortholog was found in X. campestris pv. vesicatoria, which causes disease in peppers and tomatoes  .
The XAC∆helD mutant, whose XAC2265 knocked-out gene encodes Helicase IV (HelD), showed a 100-fold smaller in planta growth than wild type strain profile (Figure 2(c)). Although it is known that HelD belongs to a group of enzymes that play a crucial role in homologous recombination and DNA repair  , it has also been recently described it as part of the transcriptional machinery in Bacillus subtilis, where it is involved in adaptation to the environment. This is because helD mutants in B. subtilis showed a reduced ability to adapt and grow when cells, in stationary phase, are diluted in rich medium  . Structurally, HelD has six conserved motifs (UvrD-helicase, UvrD_C, UvrD_C_2, AAA_19, AAA_30, AAA_12), all related to replication, recombination and DNA repair  . In bacteria of the genus Brucella, in vivo experiments on goat hosts demonstrated that mutant bacteria for an UvrD-helicase showed reduced virulence ability to attack the animal host. The authors attribute this reduced virulence phenotype to the oxidative conditions encountered by the bacteria within the host defense phagosome cells, where the absence of UvrD undermines bacterial DNA repair system when subjected to oxidative stress during host defense to pathogen infection  . During Xac attack to citrus host an analogous situation is established, where an oxidative stress due to plant resistance response against pathogen attack is also induced, suggesting a similar virulence role to Xac’s HelD as detoxifying protein. It is worth noting that orthologs of HelD with a high degree of sequence homology were observed in only two other species of Xanthomonas capable of infecting citrus, X. axonopodis Xac29-1  and X. citri subsp. citri Aw12879  , which could indicate a putative speciation of this gene in these organisms.
The in planta curve of ∆XAC3278 mutant evidenced a 4000 times smaller growth when compared with the wild type strain, clearly demonstrating that the mutation compromised the virulence and fitness of this strain within the plant (Figure 2(d)). The knock out gene for this mutant is only present in the genus Xanthomonas, since sequence homology analysis revealed only four orthologous genes of the genus Xanthomonas: X. axonopodis Xac29-1 (XAC29_16710), X. citri subsp. citri Aw12879 (XCAW_ 01591), X. fuscans subsp. fuscans (XFF4834R_chr22200) and X. alfalfae subsp. citrumelonis (XACM_2229). All four orthologous genes share the BLUF (sensors of Blue Light Using FAD) domain, which is present in several proteins (mostly bacterial) and is involved in the repression of photosynthesis genes in response to blue light (possibly oxide-reduction reactions) using flavin adenine dinucleotide (FAD) as cofactor  . BLUF domain is similar to cryptochromes, which are blue light photoreceptors involved in stem growth, cotyledon expansion, and flowering induction in plants as well as in biological clock synchronization (circadian rhythms) in animals, and are encoded by Cry1 and Cry2 genes in plants   . Genes containing the BLUF domain have been shown to be involved in functions related to survival and virulence of Xac during host plant colonization  . Interestingly, this gene is inserted into a genomic region demonstrated to result from horizontal transfer events. Apart from being acquired in these genomes, the function of this gene in these strains seems fundamental for the interaction and successful attack to plant hosts, as previously described for the XAC0068 gene.
3.2. Mutants Contributing to Partial Loss of Virulence in C. sinensis and a Total Loss in C. limonia
Five mutants (19.2%), from the twenty-six analyzed in this study, were classified into this category. None of them induced canker symptoms when inoculated in C. limonia host. However, when inoculated in C. sinensis, they caused minor necrosis (XAC∆xylR and XAC∆petA), mild hyperplasia (∆XAC1491), or minor necrosis accompanied by mild hyperplasia (XAC∆parB) (Table 2, Figure 3).
The mutant XAC∆hxylR, whose XAC0075 knocked-out gene encodes a xylose repressor-like protein (XylR), showed an approximately 100-fold smaller cell number increase in C. sinensis host, when compared to the wild type strain, demonstrating that the mutant suffered significant changes in its cellular metabolism (Figure 3(a)). An analysis using the blastx algorithm revealed that this gene is unique, with a very high sequence homology to all sequenced Xanthomonas and several Pseudomonas syringae pathovars, a causal agent of diseases in tomatoes, beans, rice, and soybeans   . In its structure, the deduced protein has a ROK domain (Table 2), which function is related to carbohydrates metabolism and transport, including glucokinase activity and possibly the production of glycoprotein compounds, such as virulence related xanthan gum    . A study investigating the promoter activities of plant-modulated bacterial gene expression revealed a PIP-BOX-homologous sequence on the XAC0075 gene  . The PIP-box or Plant-Promoter-Inducible Box is a consensus sequence of nucleotides (TTCGC...N15...TTCGC) present in the promoter region of some genes responsible for the expression of pathogenicity and virulence factors  . In X. campestris pv. campestris, a phytopathogen responsible for disease in crucifers, it was demonstrated the role of XyIR in regulating xylan synthesis and control of pathogen survival in the epiphytic phase, also participating as a regulator of genes related do pathogenic process  .
The mutant ∆XAC1491, whose knocked-out gene encodes a hypothetical protein, showed an approximately 100-fold reduced growth when compared to the wild strain, suggesting a relationship of this gene function with the Xac adaptation process inside the host plant (Figure 3(b)). Sequence similarity analysis showed that orthologs for this gene have been found only in the genomes of X. citri subsp. citri strains 306 and Aw12879 and X. axonopodis Xac29-1  . The fact that this gene is inserted in a so-called horizontal gene transfer island aids to justify its exclusivity.
The mutant XAC∆aslB which knocked out gene (XAC1927) encodes a Fe-S oxidoredutase (aslB) has showed an in planta growth 100 times lower when compared to wild type XAC306 strain (Figure 3(c)). This gene is located at a putative transfer island and was previously seem  as being expressed only during in vivo contact with citrus plant host.
The mutant XAC∆petA, whose knocked-out gene XAC2457 encodes the ubiquinol-cytochrome oxidoredutase protein (PetA), showed an approximately 1000-fold smaller growth when compared to the wild type strain inoculated in C. sinensis (Figure 3(d)). The PetA protein, along with PetB and PetC, are encoded by in tandem genes in the Xac genome (XAC2457 to XAC2455), belong to the cytochrome c-reductase complex and are directly involved in electron transfer during oxidative phosphorylation.
Figure 3. Phenotype and growth curve in vivo of mutants that led to partial loss of virulence in Citrus sinensis and total loss in Citrus limonia. In each of the five analyzed mutants ((a) to (e)), it’s possible to follow the virulence progression over the 16 days in C. sinensis (at days 4, 8, 12 and 16 after infection), while for C. limonia only the virulence phenotype at the 16 days after infection is presented. The in vivo growth curve presented is for C. sinensis.
Since oxidative phosphorylation is an energetic process common to aerobic organisms, it is evident that petA is shared by the vast majority of them  . Therefore, the loss of its functionality would result in a reduction of bacterial titration, which is clearly observed in the growth curve and, consequently, related to pathogen’s virulence.
The mutant XAC∆parB, whose knocked-out gene (XAC3906) encodes the protein for chromosome division (ParB), showed a dramatic in planta growth reduction, approximately 10,000 times, when compared to the wild type strain (Figure 3(e)). ParB and ParA protein functions are related to plasmid partitioning during cell division. The absence of these genes in Caulobacter crescentus inhibited the molecular processes responsible for the early events of cellular division  . Besides that, virulence factors can be hosted on pathogenic bacteria plasmids  , and indeed that’s the case for Xac where functional partitioning of plasmids containing virulence genes can be determinant to successful pathogen attack to host. Based on gene expression techniques, the ParB protein was shown to be induced in Xac after 12 hours growth in a XAM1 pathogenicity-promoting medium  , revealing its importance in this process.
3.3. Mutants Leading to a Partial Reduction in Necrosis and Total in Hyperplasia in C. sinensis and C. limonia
Five mutants (19.2%) (XAC∆ftsB, XAC∆yagT, XAC∆gumI, ∆XAC3368 and ∆XAC1531) were unable to induce hyperplasia and lead to a reduced necrosis in both hosts evaluated (C. sinensis and C. limonia) (Table 2, Figure 4).
The XAC∆ftsB mutant (Figure 4(a)) was knocked out for the gene XAC1720, whose functional protein corresponds to a protein involved in cellular division (FtsB). This protein, together with FtsL (XAC0773) and FtsQ (XAC0782) are small membrane proteins that create a macromolecular complex capable to promote the coordinated invagination of the plasma membrane and peptidoglycan wall and, therefore, lead to the construction of the cellular division septum  . The blastx algorithm-based analysis  showed that the XAC1720 gene has orthologs with high sequence identity (99% to 88%) only within the genera Xylella, Xanthomonas and Stenotrophomonas, which correspond to phytopathogens or opportunistic human pathogens gamma-proteobacteria  .
For ∆XAC3368 mutant (Figure 4(b)) the knocked-out gene is designated as hypothetical. This gene is showed as being specific to the genus Xanthomonas. Although hypothetical, the XAC3368 gene has two domains, the ATPase_gene1 that encodes for a small peptide (about 100 amino acids) with long hydrophobic tails found in several ba- cterial genomes and that possibly have an enzymatic function, and the Peptidase_M23, a Gly-Gly endopeptidase, which also has an enzyme function  .
The XAC∆yagT mutant (Figure 4(c)) was knocked out for XAC1189 gene, whose functional protein is predicted to be a water-soluble ferredoxin protein associated to iron-sulfur (2Fe-2S) accumulations and an important role during electron transfer in several enzymatic reactions, including the carrying of electrons during photosynthesis in bacteria, seaweed and plants   . Orthologs of the XAC1189 gene, with a high homology, were found in a large number of proteobacteria (gamma, beta, delta and alpha). However, almost a complete sequence identity was observed in the genus Xanthomonas.
Figure 4. Phenotype of mutants that led to partial loss of necrosis and total loss of hyperplasia in Citrus sinensis and Citrus limonia. In C. sinensis virulence symptoms at days 4 and 16 after infection are presented, while in C. limonia only the virulence phenotype at the 16 days after infection is presented.
The ∆XAC1531 (Figure 4(d)) mutant is knocked out in a hypothetical gene specific to genus Xanthomonas and contain the unknown function UPF0029 and DUF1949 domains (Table 2). Despite of inducing a necrotic lesion at the site of infection this mutant is not able to induce hyperplasia on infected plant leaf tissue.
The XAC∆gumI mutant (Figure 4(e)) with gene XAC2578 knocked out is responsible for encoding the GumI protein. This gene belongs to a group of 14 genes involved in the synthesis of xanthan gum, an extracellular polysaccharide (EPS) related to induced watersoaking and hyperplasia phenotype during host tissue colonization by Xanthomonas  . Xanthan gum is a virulence factor, since EPS-deficient mutants are usually avirulent    .
3.4. Mutants Contributing to a Reduction in Hyperplasia and/or Necrosis in C. sinensis and C. limonia
The XAC∆rnk mutant (Figure 5(a)) has the gene XAC0903 knocked-out, which is responsible for encoding a nucleoside diphosphate kinase (Ndk) regulator. Orthologs for rnk of Xac have only been found in bacteria of the genus Xanthomonas. Ndk is known to be involved in bacterial pathogen adaptation to host during infection  .
The XAC∆mpd mutant (Figure 5(b)) had the XAC0726 gene, responsible for encoding the methyl parathion hydrolase, knocked out. Similarity analysis revealed that this gene is highly specific, since orthologs have been found only in X. axonopodis Xac29-1, X. citri subsb citri Aw12879 and X. fuscans subsp. fuscans. The methyl parathion hydrolase (MPH) protein is an enzyme that catalyzes the degradation of a wide range of organophosphate compounds, among them the methyl parathion, which is one of the most commonly used organophosphate fertilizers worldwide, especially in fruit and vegetable farming  .
The ∆XAC1008 mutant (Figure 5(c)), which is responsible for coding a hypothetical protein, knocked out. Similarity analysis demonstrated that this gene has orthologs only in bacteria of the genus Xanthomonas and Stenotrophomonas  . It has a hydrolase_2 domain that, in B. subtilis, has been shown to be involved in cell wall hydrolysis  . Studies on genetic expression analyses showed that this protein is induced in Xac after 3 days of being inoculated into the plant  , highlighting its importance in the pathogenic process.
The XAC∆recR mutant (Figure 5(d)) was knocked-out for the gene XAC1111, which is responsible for encoding the Recombination protein RecR, which is involved
Figure 5. Mutants that led to decrease of hyperplasia and/or necrosis in Citrus sinensis and Citrus limonia. In C. sinensis virulence symptoms at days 4 and 16 after infection are presented, while in C. limonia only the virulence phenotype at the 16 day after infection is presented.
in DNA repair and replication. Similarity analysis demonstrated that this gene has orthologs only in the genera Xylella, Xanthomonas, Stenotrophomonas and Pseudoxanthomonas belonging to the family Xanthomonadaceae.
The XAC∆cynX mutant (Figure 5(e)) was knocked-out for the gene XAC2234, whi- ch encodes the MFS transporter protein, a membrane protein found in all organisms as transporter (entry and exit) of various substrates, such as sugars, drugs, metabolites, oligosaccharides and amino acids  . Nevertheless, similarity analysis showed that the XAC2234 gene is the most specific among all the mutants assessed in this study. We found only one ortholog in X. axonopodis Xac29-1. Gene expression techniques demonstrated that this protein is repressed in X. citri after 24 hours growth in a XAM1 pathogenicity-inducing medium  , suggesting a role for this protein in the early phytopathogenic process.
3.5. Mutants Contributing to Increased Necrosis in C. sinensis and C. limonia
Seven mutants (26.9%) (XAC∆orf8, XAC∆pepB, XAC∆metE, ∆XAC2344, ∆XAC3777, ∆XAC3815 and ∆XAC4321) showed an increased capability to induce plant necrosis (Table 2 and Figure 6). Knowing that the necrosis process induced in infected plant tissue is one of the defense mechanisms that the plant uses to isolate and kill pathogens   , we can infer that perhaps these genes play a role in the initial process of pathogenicity by conferring a “camouflage” to the pathogen. Therefore, the absence of translated genes would enable the plant to easily recognize the aggressor and inhibit its development and propagation through the general plant resistance response.
The XAC∆orf8 mutant (Figure 6(a)) was knocked-out for the gene XAC2243, which is responsible for encoding a protein with 86% identity (blastp analysis) to a P. aeruginosa dead box RNA helicase, which has recently characterized as being related to an indirectly up regulation of virulence type III secretion system  . This gene presents many orthologs with great similarity within the gamma/beta/alphaproteobacteria. However, among the bacteria of the genus Xanthomonas, an ortholog was only found in X. axonopodis Xac29-1, X. citri subsp. citri Aw12879, X. campestris pv. campestris B100 and X. campestris pv. vesicatoria, which could indicate a horizontal transfer of this gene.
The genes deleted in the mutants ∆XAC2344 (Figure 6(b)), ∆XAC3777 (Figure 6(c)), ∆XAC3815 (Figure 6(d)) and ∆XAC4321 (Figure 6(e)), encode for hypothetical genes. All genes had orthologs only in bacteria of the genus Xanthomonas. The gene XAC2344 has an YCII domain, which might indicate that this gene encodes for a protein with an enzymatic function, since it has highly conserved histidine and aspartate regions  . Studies on genetic expression analyses showed that this protein is induced in wild type strains of Xac immediately after the first 24 hours upon inoculation  , showing that this protein plays a role in the pathogenic process. The gene XAC3777 has a nucleoplasmin domain, which is involved in the process of chromatin decondensation  . The deduced protein from gene XAC3815 has 3 domains (DUF3667, CitX, and zf-C3HC4_3), which are still not well characterized. Finally, the gene XAC4321 has no identified domains so far, thus making it impossible to infer any function of the protein
Figure 6. Mutants that led to increased necrosis in Citrus sinensis and Citrus limonia. In C. sinensis virulence symptoms at days 4 and 16 after infection are presented, while in C. limonia only the virulence phenotype at the 16 days after infection is presented.
encoded by this gene. In X. vesicatoria this gene was annotated as a putative secreted protein.
The XAC∆pepB mutant (Figure 6(f)) was knocked-out for the gene XAC3987, which is responsible for encoding leucine aminopeptidase, an enzyme that catalyzes the removal of leucine residues from the N-terminus of proteins  . Similarity blastp analysis demonstrated that all representatives of the genera Xylella, Xanthomonas, Stenotrophomonas and Pseudoxanthomonas have an ortholog for this gene.
The XAC∆metE mutant (Figure 6(g)) was knocked-out for the gene XAC0336, which is responsible for encoding the 5-methyl-tetrahydropteroyltriglutamate-homocysteine S- methyltransferase, a protein involved in cysteine and methionine amino acid metabolism, acting as a methyltransferase in the conversion of L-homocysteine into L-methionine  . It was shown that this gene is conserved in a large number of gamma/beta-pro- teobacteria. In a recent study with Ralstonia solanacearum, it was observed that both the MetR, a classic repressor of genes involved in methionine synthesis, and HrpG, a regulator associated with the expression of T3SS gene effectors, are capable of modulating the expression of MetE  , which highlights the importance of this gene in processes of plant adaptation and virulence induction.
3.6. Analysis of Mutated Orthologous Genes in Other Genomes of the Family Xanthomonadaceae
In an attempt to understand the possible origin of these genes in the Xac genomes, and the possible presence of homologs in other bacteria of the same family, the presence or absence of orthologs was assessed by sequence comparison. Another thirteen different species of bacteria of the genus Xanthomonas were included in this analysis, four species of the genus Stenotrophomonas, five of the genus Xylella and two of Pseudoxanthomonas (see list and abbreviations in the methodology section), all with their respective genomes deposited in the Kegg (Figure 7). As a result, four sets of genes were identified in a cladogram generated from a presence/absence matrix.
From the first set consisted of six mutants, which genes were characterized as being unique to bacteria strains of the genus Xanthomonas that infect citrus (13-XAC∆aslB, 16-XAC∆helD, 1-∆XAC0068, 14-XAC∆cynX, 10-∆XAC1491 and 20-∆XAC3278), three of which show a complete absence of symptoms when inoculated into both hosts analyzed, 6, 1 and 20. Mutant 20 still stands out in this set for being one of the 3 mutants whose genes are unique to the species associated with citrus, and as for mutants 13 and 10, which are inserted in into a putative horizontal transfer islands. This was the only gene associated with the species that infect citrus, which was also found in X. axonopodis pv. citrumelo F1. The gene cynX was found only in Xac and Xao, and XAC1491 only in Xac and Xci.
In the second set, 7 other mutants were grouped together (23-∆XAC3815, 26-∆XAC4321, 5-∆XAC0732, 9-XAC∆yagT, 22-∆XAC3777, 2-XAC∆xylR and 19-XAC∆gumI). This set draws our attention, since their respective knocked-out genes were characterized as being unique to the genomes of bacteria of the genus Xanthomonas, except for mutants 23 and 19, whose genes were also found in bacteria of the genus Pseudoxanthomonas. Also in this set, it is important to highlight that subgroups can be observed, since there is the absence of genes shared by specific species, as it is the case for the genes knocked- out in mutants 23, 26 and 5, absent in bacteria of the strain X. campestris, and in mutants 9 and 22, absent in bacteria of the strain X. oryzae. In this set of knocked-out genes, the only ortholog present in X. albilineans is the XAC4321 gene.
The third set of 6 knocked-out genes were also highlighted (21-∆XAC3368, 6-XAC∆rnk, 3-XAC∆metE, 17-∆XAC2344, 11-∆XAC1531 and 7-∆XAC1008). In this case, it is due to the fact that they coexist only in bacteria of the genera Xanthomonas and Stenotrophomonas, except for the genes related to mutants 11, 7 and 6, which also coexist in bacteria of the genus Pseudoxanthomonas. Only the genes associated to mutants 21 and 6 were not found in the genomes of X. albilineans and X. oryzae pv. oryzicola BLS256.
Figure 7. Presence and absence analysis of mutated genes in other bacteria of the family Xanthomonadaceae. The green color shows the presence of orthologous genes in their respective XAC mutants analyzed in this study and named according to Figure 1(c). The organisms are presented in a horizontal cladogram based on a gene matrix of presence and absence enabling the creation of the cladogram. The phylogeny of the strains based on conserved gene sequences (gyrB or 16S-rDNA) and maintained by the displacement of two clads are highlighted by full circles, which should be allocated in the positions of empty circles of the same color. The red triangles highlight the genes inserted in the genomic islands. The yellow arrows indicate nonpathogenic mutants in both hosts analyzed, whereas white arrows show a total loss of virulence only in Citrus limonia. Red outlines highlight specific genes in strains associated with citrus. Yellow outlines, specific genes in bacteria of the genus Xanthomonas, however, absent in some specific strains set. The numbers from 1 to 4 on the right side of the design highlight determined sets of genes discussed in the text. Blue outlines, specific genes of bacteria of the genus Xanthomonas and in some cases, present also in Pseudoxanthomonas. White outlines, total absence of genes in Xylella.
Contrary to what has been described for the three sets analyzed so far, none of these related genes was found in the genomes of bacteria of the genus Xylella. This event might have a direct relationship with the alleged massive genome reduction that occurred in the species of this genus  . However, it can also result from the acquisition of other genera in the genomes.
Finally, a fourth set of genes was found consensually in the genomes of all species (24-XAC∆parB, 25-XAC∆pepB, 18-XAC∆petA, 12-XAC∆ftsBand 8-XAC∆recR). The products of these genes are associated with fundamental cellular processes such as DNA replication and repair, cell growth and development. Although essential to the maintenance of cellular survival, their absence did not cause any reduction in their growth rates in vitro.
The data presented in this study enabled to associate twenty-six Xac genes, where eleven were hypothetical, with adaptation events and pathogenicity in compatible hosts, e.g. C. sinensis and C. limonia. Six of these genes were characterized as being unique to species infecting citrus, including X. citri subsp. citri Aw12879, X. axonopodis Xac29-1 and X. axonopodis pv. citrumelonis F1. Three of the six citrus species associated with genes are inserted into putative horizontal transfer islands. Other seven knocked out genes are specific to the genus Xanthomonas. Therefore, this in vivo study of a new repertoire of genes related to pathogenicity and virulence contributes to a better understanding of the biology of Xac-citrus interaction, allowing its extrapolation to other pathosystems and suggesting new targets to further studies on plant-pathogen disease.
This work has been supported by Fundação de Amparo à Pesquisa do Estado de São Paulo―FAPESP, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior― CAPES, CNPq and by Fundo de Defesa da Citricultura (FUNDECITRUS). C. B. F. is thankful to CAPES and FUNDECITRUS for receiving an MSc fellowship for the development of this work. J. C. F. O. was recipient of a Jovem Pesquisador research grant from FAPESP (Proc. 04/02006-7). J. A. F. and M. I. T. F. are recipients of research fellowships from CNPq. This work is part of the MSc thesis of C. B. F.
*These authors contributed equally to this work.