An Extracytoplasmic Function Sigma Factor Required for Full Virulence in Xanthomonas citri pv. citri

ABSTRACT

The genus Xanthomonas includes more than 30 phytopathogenic species that infect a wide range of plants and cause severe diseases that greatly impact crop productivity. These bacteria are highly adapted to the soil and plant environment, being found in decaying material, as epiphytes, and colonizing the plant mesophyll. Signal transduction mechanisms involved in the responses of Xanthomonas to environmental changes are still poorly characterized. Xanthomonad genomes typically encode several representatives of the extracytoplasmic function σ (σ^ECF) factors, whose physiological roles remain elusive. In this work, we functionally characterized the Xanthomonas citri pv. citri EcfL, a σ^ECF factor homologous to members of the iron-responsive FecI-like group. We show that EcfL is not required or induced during iron starvation, despite presenting the common features of other FecI-like σ^ECF factors. EcfL positively regulates one operon composed of three genes that encode a TonB-dependent receptor involved in cell surface signaling, an acid phosphatase, and a lectin-domain containing protein. Furthermore, we demonstrate that EcfL is required for full virulence in citrus, and its regulon is induced inside the plant mesophyll and in response to acid stress. Together, our study suggests a role for EcfL in the adaptation of X. citri to the plant environment, in this way contributing to its ability to cause citrus canker disease.

IMPORTANCE The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σ^ECF). Here, we describe a new σ^ECF factor found in several Xanthomonas species, demonstrating its role in Xanthomonas citri virulence to citrus plants. We show that EcfL regulates a single operon containing three genes, which are also conserved in other Xanthomonas species. This study further expands our knowledge on the functions of the widespread family of σ^ECF factors in phytopathogenic bacteria.

INTRODUCTION

Bacterial fitness in distinct environments relies on the ability to finely monitor a wide diversity of environmental cues and rapidly adapt to sudden changes in the milieu, such as nutrient availability and both biotic and abiotic stresses. To this end, bacteria deploy a varied repertoire of signaling pathways that redirect gene expression upon sensing specific stimuli, including one-component systems, two-component systems, Ser/Thr protein kinases, and alternative sigma factors (1–5). In line with that notion, the abundance of genes encoding signal transduction pathways in bacterial genomes shows a positive correlation with the diversity of environments colonized by each species (2, 6).

Extracytoplasmic function σ (σ^ECF) factors form one of the most diverse and abundant signal transduction pathways in bacteria, mediating a wide range of stress responses (1). Bacterial σ factors are subunits of the RNA polymerase that recognize the −10 and −35 promoter elements and promote transcription initiation (7). The σ⁷⁰ family encompasses the primary and most alternative σ factors, and is further divided into four groups, according to the architecture of up to four functional domains (σ₁ to σ₄). σ^ECF factors, originally identified as playing roles in the extracytoplasmic stress response (8, 9), comprise σ⁷⁰ group 4 and are defined by the absence of domains σ₁ and σ₃ (10, 11). In addition, common themes pertaining to this group include (i) negative regulation of σ^ECF activity by transmembrane anti-σ factors, usually encoded in the same operon as the cognate sigma factor, (ii) positive autoregulation of the σ^ECF/anti-σ encoding operon, and (iii) recognition of conserved “AAC” and “CGT” motifs in the −35 and −10 promoter elements, respectively (8, 9). Nevertheless, comparative genomic studies and an ever-expanding repertoire of characterized σ^ECF factors have revealed a wide diversity of functions and modes of regulation of σ activity, including regulation of cytoplasmic stress responses, negative regulation by soluble anti-σ factors, presence of regulatory C-terminal extensions, and activation by transmembrane Ser/Thr kinases and two-component systems (1, 4, 5, 12). Together, data generated from comparative genomics reinforce the need for functional studies to broaden our understanding of this important group of bacterial regulators.

A limited number of σ^ECF factors have been characterized in phytopathogenic bacteria. The ECF HrpL is essential for virulence of several species, controlling the expression of type III secretion system (T3SS) genes in Pseudomonas syringae, Pectobacterium carotovorum, Dickeya dadantii, and Erwinia amylovora (13). Likewise, the σ^ECF PrhI from Ralstonia solanacearum is required for induction of T3SS genes in response to plant cell contact (14). PrhI is a member of the iron starvation-responsive FecI subgroup, which has also been characterized in P. syringae (15). FecI-like σ^ECF factors belong to subgroups ECF05 to ECF10 of the original classification scheme by Staron et al. (1), but a recent reclassification combined ECF05 to ECF09 into the new subgroup ECF243, while members of ECF10 form the subgroup ECF240 (5). fecI-like genes are typically found in operons with a gene encoding the cognate anti-σ factor (FecR-like), which contains a predicted transmembrane region (1). Several FecR-like proteins also present a pro-σ role, increasing the activity of the cognate sigma factor, which is a distinguishing feature of this family of σ regulators (16, 17). The prototypical member of ECF243 is E. coli FecI, which is involved in the regulation of Fe³⁺-citrate transport genes in response to iron starvation (18, 19). FecI is activated by a transenvelope signal system that involves the anti-σ FecR and the outer membrane TonB-dependent receptor (TBDR) FecA, which is the Fe³⁺-citrate transporter and acts on signal transduction by means of its N-terminal extension. Binding of Fe³⁺-citrate to FecA promotes the interaction between the N-terminal transducer domain of FecA and the periplasmic domain of FecR, triggering the activation of FecI, which ultimately induces the expression of the Fe³⁺-citrate transport genes fecABCDE (19). FecA-encoding genes are typically found adjacent to the fecIR operon in bacterial genomes, which is used as a prediction tool for members of this subgroup.

The genus Xanthomonas represents the largest group of phytopathogenic bacteria and causes disease in a wide variety of economically important plants, including rice, sugarcane, pepper, tomato, and citrus. In addition to colonizing different plant tissues, some species can also be found in the soil in association with dead plant material or living on the surface of leaves and twigs as epiphytes (20). As predicted by their ability to colonize distinct environmental niches, Xanthomonas spp. typically possess several σ^ECF factors. Xanthomonas citri pv. citri is the causal agent of citrus canker, a disease that severely affects all of the economically important citrus cultivars, greatly reducing crop quality and yield. The bacteria colonize the host mesophyll, producing eruptive and hyperplastic water-soaked lesions that further progress to tissue necrosis in the affected leaves, stems, and fruits (21). The genome of X. citri strain 306 encodes nine σ^ECF factors, and the sole representative of this family that has been characterized in the species is EcfK, which is required for resistance to predation by the bacterivorous amoeba Dictyostelium discoideum (22). EcfK is part of a subgroup of σ^ECF factors (ECF43) that are activated upon phosphorylation by a cognate Ser/Thr kinase (PknS) and are not found in association with typical anti-σ factors (1, 22, 23). Our previous work has shown that X. citri EcfK is required for expression of a type VI secretion system (T6SS) in response to interaction with D. discoideum and is encoded in the vicinity of the T6SS gene cluster (22). Interestingly, a gene encoding another σ^ECF factor (XAC4129), here named ecfL, is found adjacent to and in the opposite direction of transcription of ecfK, in a 14.6-kb region that separates the two gene clusters that encode the complete T6SS. The genomic context of ecfL is characteristic of the FecI group, with adjacent homologues of fecR and fecA.

In this work, we investigated the role of EcfL in several aspects of X. citri physiology, including virulence, response to nutrient starvation, and resistance to predation by amoeba. Results from transcriptome analysis showed that EcfL regulates a single operon composed of three genes, which includes a fecA homologue. We also show that EcfL is required for full virulence of X. citri in sweet orange plants and that expression of ecfL and members of its regulon is induced during growth in planta. Despite its classification as a FecI-like σ^ECF factor, ecfL is not required for the response to iron starvation, suggesting a distinct role in X. citri. Together, our results describe the first characterization of a FecI-like σ^ECF factor in the Xanthomonadales order.

RESULTS

EcfL is part of a FecI-like subgroup whose members are restricted to the Xanthomonadales order and share a conserved genomic context.

Based on the most recent classification of σ^ECF factors available at the web-based resource ECF Hub (https://www.computational.bio.uni-giessen.de/ecfhub) (5), ecfL is a member of ECF243 subgroup 48, composed of 32 homologues with distribution restricted to the Xanthomonadales, 20 of them found in species from the Xanthomonas genus. The X. citri genome also encodes another FecI-like ECF243, XAC2191, which belongs to subgroup 59, whose members are mostly found in Xanthomonadales, but also in the Burkholderiales and Sphingomonadales orders. A phylogenetic analysis including representatives from each FecI-like ECF05-ECF10 groups showed that EcfL does not group with the clade formed by well-characterized ECF243 σ factors involved in iron acquisition that includes FecI_E.coli and PvdS_P.aer, but forms a distinct clade (Fig. 1A; see also Table S1 in the supplemental material). The fact that EcfL and XAC2191 share limited sequence identity (19.9%) and similarity (33.9%) suggests that they play distinct biological roles in X. citri.

FIG 1.

Phylogenetic analysis of EcfL homologues and genome context conservation within the Xanthomonadales. (A) Phylogenetic distribution of EcfL and representatives of the FecI-like extracytoplasmic function (ECF) groups 5 to 10. Proteins are identified by their name (when applicable), Kyoto Encyclopedia of Genes and Genomes (KEGG) entry (UniProt entry for PrhI, PvdS, and PbrA) and species name. ECF classification according to Staron et al. (1) and the most recent scheme available at the ECF Hub website (5) is indicated for each sequence. Sequences of Escherichia coli and Xanthomonas citri primary σ factor RpoD are also included in the analysis. (B) Phylogeny of EcfL homologs identified in the Xanthomonadales order and schematic representation of genome context within each clade. Branches indicated by A′, A″, B and C are colored red, green, purple and black, respectively. The asterisk identifies the exception inside clade A″, since no endonuclease/exonuclease/phosphatase domain (EEP)-encoding gene is found next to the ecfL homologue. The trees were inferred by the maximum-likelihood method with 1,000 bootstrap replicates using MEGA X software (71).

A phylogenetic analysis of EcfL homologues from the Xanthomonadales order retrieved from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (24) grouped them into three major clades, with conserved genomic contexts for members of the same clade (Fig. 1B). All ecfL homologues are encoded as a predicted operon with a FecR-like anti-σ factor gene, with a FecA-like TBDR encoding gene found immediately downstream from the σ/anti-σ operon. In homologues from clade A, the fecA-like gene is found in a putative operon with genes encoding a histidine acid phosphatase and a conserved hypothetical protein with an endonuclease/exonuclease/phosphatase domain (EEP), whereas only an acid or alkaline phosphatase gene is found co-occurring with the fecA-like gene in other clades (Fig. 1B).

EcfL controls a small regulon formed by a single adjacent operon.

To identify genes regulated by EcfL in X. citri, we analyzed the transcriptome of a strain overexpressing ecfL (wild type [WT]/ecfL⁺⁺) by transcriptome sequencing (RNA-Seq). For this analysis, we cloned ecfL under the control of an arabinose-inducible promoter in a multicopy plasmid (pBRA). Interestingly, overexpression of EcfL by addition of arabinose to the culture medium completely inhibited the growth of X. citri, without significantly reducing bacterial viability, as determined by CFU counting (Fig. 2A and B). This bacteriostatic effect was not observed in strains overexpressing the σ^ECF factor EcfK or the constitutively active version EcfK^T51E (Fig. 2A) (22, 23). We also analyzed bacterial membrane integrity and cell morphology by fluorescence microscopy, after differential staining with the commercial Live/Dead BacLight kit, which uses SYTO9 (green) and propidium iodide (PI; red) to stain live and dead/membrane-permeabilized cells, respectively (25). Results from this analysis showed that ecfL overexpression does not affect cell morphology, but caused a moderate increase in the number of PI-positive (PI⁺) cells (Fig. S1). Results from CFU counting after 8 h of incubation with the inducer did not show a decrease in the number of viable cells upon ecfL overexpression (Fig. 2B), suggesting that the increase in PI⁺ cells may be caused by changes in homeostasis of the extracytoplasmic compartment and inner and/or outer membrane permeabilization.

FIG 2.

EcfL regulates three genes in the vicinity of its locus. (A and B) Overexpression of ecfL has a bacteriostatic effect in X. citri. (A) Growth curve of strains carrying constructs for overexpression of ecfL (WT/ecfL⁺⁺), ecfK (WT/ecfK⁺⁺), and the constitutively active version ecfK^T51E (WT/ecfK^T51E) under the control of the arabinose-inducible promoter pBAD. The wild-type strain carrying the empty vector was used as a control. Results are the mean of three biological replicates ± standard error. (B) Analysis of bacterial viability upon overexpression of ecfL by determination of CFU/mL. Samples were harvested after 8 h of growth in the presence of glucose or arabinose for determination of CFU/mL. Samples for the 0-h time point were obtained immediately after dilution of saturated bacterial cultures. Results are the mean of three independent experiments (n = 3) ± standard error. Statistical analysis was performed using Student’s t test; *, P < 0.05; **, P < 0.01; ns, nonsignificant. (C) Identification of EcfL-regulated genes by differential expression analysis (RNA-Seq) in response to overexpression of ecfL in a multicopy plasmid (WT/ecfL⁺⁺) compared to expression in the WT strain with empty vector. Results were obtained from two independent biological replicates. Genes that did not show significant changes in expression levels are indicated by black dots. Differentially expressed genes (log₂ fold change ≥ 1 or ≤ −1) with statistical significance (adjusted P value < 0.01) are depicted in red dots. (D) Validation of the RNA-Seq results by qRT-PCR using independent samples. The results are shown as relative expression levels in strain WT/ecfL⁺⁺ compared to the WT with empty vector. The housekeeping gene rpoB was used for normalization. Results are the mean of three biological replicates (n = 3) ± standard error.

To overcome this effect and avoid pleiotropic consequences of EcfL overproduction, we performed RNA-Seq analysis using exponential-phase cultures grown in the absence of the inducer, since this condition is sufficient to promote a greater than 900-fold increase in ecfL mRNA levels, as determined by quantitative real-time PCR (qRT-PCR) (Fig. S2). The following three genes were significantly induced upon overexpression of ecfL: XAC4131 (fecA-like), XAC4132, and XAC4133 (Fig. 2C). A moderate increase in the mRNA levels of XAC4130, which encodes the putative cognate anti-σ, was detected in our analysis (log₂ fold change = 1.6), but such an increase resulted from reads mapping to the 5′ region of XAC4130 included in the ecfL overexpression construct (Fig. S3). Genes downregulated in the WT/ecfL⁺⁺ strain were not identified in our analysis (Fig. 2C). RNA-Seq results were validated by qRT-PCR with independent biological samples, which showed a similar pattern of induction for all three genes, with XAC4131 displaying the most pronounced fold change in mRNA levels (Fig. 2D).

The genome context is suggestive of an operon structure formed by XAC4131, XAC4132, and XAC4133, since the distance between each of them is less than 100 bp (Fig. 3A). To test whether XAC4131-XAC4132-XAC4133 forms a single transcriptional unit, we performed RT-PCR using primer pairs that anneal in distinct adjacent genes to amplify from reverse-transcribed mRNA from the strain WT/ecfL⁺⁺. Results from this analysis confirmed that XAC4131, XAC4132 and XAC4133 are expressed as one transcript (Fig. 3A), showing that EcfL regulates a single operon that is immediately downstream of its coding gene. In order to identify the promoter sequence recognized by EcfL, we determined the XAC4131 transcription start site (TSS) by 5′ rapid amplification of cDNA ends (RACE), using mRNA samples from the WT/ecfL⁺⁺ strain. The experimentally identified TSS is located 22 bp upstream from a predicted start codon, with an associated Shine-Dalgarno motif (Fig. 3B). The upstream sequences (from −50 to the start codon) of fecA-like representatives found associated with ecfL homologues (species from the phylogenetic analysis described in Fig. 1B) were scanned for the presence of common motifs using the MEME tool from MEME Suite (https://meme-suite.org/meme/) (26, 27). A conserved motif of 35 nucleotides was identified in 13 out of the 26 sequences analyzed, which corresponded to fecA-like promoters for cognate EcfL homologues from clade A′ (to which ecfL belongs) and clade B (ecfL of Stenotrophomonas spp.) (Fig. 3B). Our results provide experimental evidence for a consensus promoter motif for ECF248s48 members. This motif corresponds well to the consensus predicted for the ECF243s48 subgroup by bioinformatic studies (Fig. 3B) (5).

FIG 3.

Operon structure analysis of EcfL-regulated genes and identification of the EcfL consensus promoter. (A) The EcfL regulon comprises a single transcriptional unit. RT-PCR analysis using cDNA samples from the WT/ecfL++ strain and primer pairs annealing in distinct adjacent genes, as indicated in the schematic representation of the ecfL-XAC4130-XAC4131-XAC4132-XAC4133 genome region. The amplicons are indicated by I to V in the scheme. Amplicons I and II are generated from a template corresponding to transcription of adjacent genes into a single mRNA and confirm the operon structure of XAC4131, XAC4132, and XAC1433. Amplicons III, IV, and V correspond to each individual gene and were included as a control of mRNA integrity. C−, total mRNA sample; S, cDNA test sample; C+, X. citri genomic DNA; C−*, no-template control. (B) Determination of the transcription start site (TSS) of XAC4131 by 5′ rapid amplification of cDNA ends (RACE). The sequence chromatogram of a representative clone from a total of five is shown. A consensus promoter motif was identified based on a search among the upstream sequences of fecA-like homologues using MEME software, and corresponds to the −10 and −35 regions relative to the transcription start site (TSS) of XAC4131. The XAC4131 upstream sequence is depicted above the chromatogram, with −10 and −35 promoter sequences in red and aligned to the predicted consensus for the ECF243 subgroup 48 (5). The consensus motif logo and an alignment with upstream sequences of representative homologues of XAC4131 is shown below the chromatogram. Buj, S. maltophilia JV3; Sten, Stenotrophomonas sp. WZN-1; Xci, X. citri subsp. citri Aw12879; xac, X. citri pv. citri 306; xph, X. phaseoli pv. phaseoli CFBP6546R; xpe, X. perforans LH3.

XAC4130 encodes a FecR-like protein that interacts with EcfL.

The ecfL-XAC4130 gene pair has overlapping stop and start codons, which is suggestive of an operon structure with translational coupling (28). Similarly to FecR-like anti-σ factors, the protein encoded by XAC4130 contains one transmembrane helix and a periplasmic C-terminal region that possesses a FecR domain (Pfam family PF04773) (29) linked to a DUF4974 region, which contains a LLLV motif required for FecR interaction with the TBDR FecA in Escherichia coli (Fig. 4A) (30). The amino-terminal cytoplasmic domain of FecR-like proteins directly interacts with the cognate σ factor and typically contains a DUF4880 (Pfam family PF16220) with three conserved tryptophan residues, with one of them (Trp40) forming part of the TrpX₃AspX₂His motif (31, 32). This region is characterized by the presence of a structural anti-σ domain (ASD) conserved among ECF anti-σ factors, which folds into a 3-helix core bundle (31, 33). Protein sequence alignment and secondary structure prediction of XAC4130 cytoplasmic domain (residues 1 to 115) revealed the presence of the three helices, in addition to the presence of DUF4880 and the conservation of the tryptophan residues and the TrpX₃AspX₂His motif, with a substitution to TrpX₃SerX₂His that is also found in other homologues (Fig. S4). We tested the direct interaction between the cytoplasmic domain of XAC4130 (residues 1 to 117, XAC4130_1-117) and EcfL using the yeast two-hybrid system. Our results show that the cytoplasmic domain of XAC4130 is sufficient for the interaction with EcfL (Fig. 4B). Therefore, XAC4130 possesses the characteristics of a canonical FecR-like protein and contains a conserved ASD in its cytoplasmic region that is sufficient for interaction with the cognate σ factor, as shown in homologous systems, such as FecIR and PupIR (33, 34).

FIG 4.

XAC4130 encodes a typical FecR-like protein. (A) Domain organization of XAC4130 (SMART accession number s|190486.XAC4130). Conserved amino acids that form the hydrophobic core (LLLV) located inside the DUF4974 are highlighted (I278, L285, F292, H301, L316, and R325). DUF, domain with unknown function; TM, transmembrane region. (B) Yeast two-hybrid assays showing the interaction between EcfL and the cytoplasmic domain of the anti-sigma factor XAC4130. The region encoding the cytoplasmic domain of XAC4130 (residues 1 to 117) and the gene ecfL (xac4129) were cloned in vectors pOAD (LEU2+) and pOBD (TRP1+), respectively. Growth in medium without tryptophan (Trp⁻) or leucine (Leu⁻) are shown as controls for the presence of pOBD and pOAD constructs, respectively. Growth in the absence of tryptophan, leucine, histidine, and adenine supplementation indicates a productive interaction and consequent activation of HIS3 and ADE2 reporter genes. (C) Expression of ecfL, XAC4131, XAC4132, and XAC4133 is not significantly affected by deletion of XAC4130. Total mRNA was obtained from exponential-phase cultures of WT and Δ4130 strains and used for relative expression analysis by quantitative real-time PCR (qRT-PCR), with the WT strain as reference. The housekeeping gene rpoB was used for data normalization. (D) Overexpression of XAC4130 induces expression of EcfL-regulated genes. qRT-PCR analysis of mRNA levels of XAC4131 and XAC4132 in strain Δ4130 carrying XAC4130 in the pBRA vector, after induction for 30 min with arabinose. The Δ4130 strain with empty vector cultivated under the same conditions was used as reference for relative expression and rpoC was used for data normalization. Results are the mean of three biological replicates ± standard error.

To test for a possible inhibitory effect of XAC4130 on EcfL activity, we generated a XAC4130 deletion (Δ4130) strain and analyzed the mRNA levels of ecfL and EcfL-regulated genes, relative to those of the parental strain (WT). This analysis showed that ecfL, XAC4131, XAC4132, and XAC4133 are not differentially expressed in the absence of XAC4130 (Fig. 4C). Based on a model of regulation of EcfL activity solely by anti-σ inhibition, an induction of EcfL-regulated genes was expected to occur in the Δ4130 strain. We also assessed the putative anti-σ activity of XAC4130 by overexpressing the gene from the arabinose-inducible promoter on the pBRA vector, followed by analysis of expression of EcfL target genes. Results from qRT-PCR analysis showed a 2-fold increase in mRNA levels of XAC4131 and XAC4132 in the Δ4130 strain overexpressing XAC4130 compared to those in the strain with empty vector (Fig. 4D). This result indicates that XAC4130 does not have an inhibitory effect on EcfL under these experimental conditions and provide evidence for its pro-σ activity, promoting the transcription of EcfL target genes, as previously described for other FecR-like homologues (16, 17).

EcfL is not involved in iron or phosphate starvation responses and does not engage in a regulatory cascade with EcfK.

To further characterize the role of EcfL in X. citri, we generated an in-frame deletion strain, the ΔecfL strain. Since EcfL is a member of the major family of ECFs involved in iron acquisition, we tested whether it plays a role in the response of X. citri to iron starvation, by addition of the iron chelator 2,2′-dipyridyl to bacterial cultures growing in XVM2 synthetic medium (35). Our results show that deletion of ecfL does not affect the growth of X. citri under iron-limiting conditions (Fig. 5A). Moreover, results from qRT-PCR analysis showed that ecfL, XAC4131, and XAC4132 are not induced in response to iron depletion (Fig. S5).

FIG 5.

EcfL is not required for X. citri response to iron and phosphate starvation. (A) Growth curve of wild-type (WT) and ΔecfL strains under iron starvation by addition of the iron chelator 2,2′-dipyridil. Results are the mean of at least five independent experiments ± standard error bars. (B) Growth curve of WT and ΔecfL in phosphate replete M4 medium (M4), in M4 low-phosphate medium (LPM4), or M4 low-phosphate medium supplemented with phytic acid as the phosphate source (LPM4 + PA). The results are the mean of four independent experiments ± standard error bars.

Xanthomonas genomes typically have an overrepresentation of genes encoding TonB-dependent receptors, ranging from 30 to more than 60 genes, whereas the vast majority of Gram-negative bacteria present up to 14 TBDRs (36). These outer membrane receptors are commonly involved in the uptake of iron-siderophore complexes and vitamin B₁₂, but a role in the acquisition of other nutrients is suggested in bacteria with a greater number of these receptors, as shown for the carbohydrate scavenging loci in Xanthomonas campestris (36). Coexpression of the TBDR gene XAC4131 with XAC4132, which encodes an acid phosphatase similar to phytases, led us to investigate whether the EcfL regulon is required for the response to phosphate starvation and bacterial acquisition of phytic acid (myo-inositol hexaphosphate), the main phosphorus source found in soil and plants (37, 38). The X. citri WT and ΔecfL strains grew indistinguishably in replete M4 minimal medium containing high phosphate concentrations (M4; 60 mM K₂HPO₄ plus 30 mM KH₂PO₄) (Fig. 5B). A low-phosphate M4 medium (LPM4), with a 30-fold reduction in concentrations of phosphate sources, did not support the growth of either X. citri strain. Supplementation of LPM4 with phytic acid (3 mM) supported the growth of the X. citri WT strain, showing that the bacterium is able to use phytic acid as a phosphate source (Fig. 5B). Deletion of ecfL caused only a discrete reduction in bacterial growth rate compared to that of the WT, showing that the ability to utilize phytic acid is retained in the mutant strain (Fig. 5B). These results suggest that the EcfL regulon is not required for growth of X. citri when phytic acid is the sole phosphate source, although the phytase encoded by XAC4132 possibly contributes to this function.

The X. citri ecfL and homologues from highly related species that form clade A′ (Fig. 1B) are located in a genomic region that separates the two gene clusters encoding a fully functional T6SS (Fig. 6A) (22). In addition, another σ^ECF factor, ecfK, that regulates the T6SS, is located in the opposite direction of ecfL. Therefore, we hypothesized that EcfL might be involved in a regulatory cascade involving two distinct σ^ECF factors to regulate T6SS-related functions, e.g., T6SS effectors. In line with this hypothesis, a secreted phytase is important for resistance to amoeba predation in Legionella pneumophila (39). Results from our transcriptome analysis ruled out a role of EcfL in the regulation of ecfK and T6SS gene clusters (Fig. 2C). We then analyzed whether overexpression of a constitutively active version of ecfK, ecfK^T51E, affects ecfL mRNA levels, using qRT-PCR. Overexpression of ecfK^T51E induced transcription of the T6SS gene vgrG, as we previously described (22, 39), but the mRNA levels of ecfL were similar to those in the control strain carrying the empty vector (Fig. 6B). In addition, phagocytic plaque assays to test for resistance to D. discoideum predation showed that the ΔecfL and ΔXAC4130 strains are indistinguishable from the parental strain, while the T6SS mutant strain used as a control was significantly more sensitive to amoeba predation, as previously described (Fig. 6C). The genes ecfL, XAC4131, XAC4132, and XAC4133 were not induced in response to coincubation with D. discoideum for up to 24 h, as analyzed by qRT-PCR (data not shown), a condition shown to induce T6SS gene expression (22). Based on these results, we ruled out a role for EcfL in the regulation of genes associated with T6SS function in X. citri.

FIG 6.

EcfL is not involved in the EcfK signaling cascade that is required for resistance to Dictyostelium discoideum predation. (A) Schematic representation of ecfL genome context, depicting its location in the type VI secretion system (T6SS) interspacing region and highlighting the adjacent genes coding for the T6SS regulator EcfK and its cognate Ser/Thr kinase PknS. Location of the depicted genome region relative to the X. citri chromosome is indicated. (B) Overexpression of a constitutively active EcfK version (ecfK^T51E) does not induce ecfL transcription. qRT-PCR analysis of ecfL mRNA levels in the wild-type strain carrying a copy of ecfK^T51E under the control of the arabinose inducible promoter pBAD in the pBRA vector (WT ecfK^T51E). The vgrG gene was also included in the analysis for comparison, as it is induced by expression of ecfK^T51E, as described previously (21). The WT strain carrying the empty vector was used as the reference for relative expression analysis. Samples were from mid-logarithmic-phase cultures induced for 30 min with arabinose. Results are the mean of two independent experiments ± standard error bars. (C) Resistance of WT, ΔecfL, and Δ4130 strains to phagocytosis by D. discoideum as assessed by phagocytic plaque assay. The T6SS in-frame deletion mutant Δhcp was included for comparison, showing sensitivity to amoeba predation as previously described (22, 68). Representative experiment from at least three biological replicates.

EcfL is required for full virulence of X. citri and is induced in planta and by acidic pH.

We investigated the role of EcfL in X. citri virulence in infection assays in the susceptible host Citrus sinensis (sweet orange) using the ΔecfL and Δ4130 deletion strains. The ΔecfL strain is severely impaired in disease progression, showing reduced canker lesions, compared to the WT and Δ4130 strains (Fig. 7A). Moreover, the phenotype was fully restored by complementation with the wild-type copy of ecfL in the pBRA vector. Bacterial inoculation was performed by the pinprick method in experiments shown in Fig. 7A, and similar results were obtained when bacterial cultures were directly inoculated inside the plant mesophyll by syringe infiltration (Fig. S6A). We also analyzed the expression of ecfL and its regulon during bacterial growth inside susceptible host plants by qRT-PCR. Expression of ecfL was induced 6 h post-inoculation (hpi) and showed greatest expression levels at 48 and 72 hpi. A similar expression pattern was observed for XAC4131, XAC4132, and XAC4133, which showed increased mRNA levels from 24 to 72 hpi. (Fig. 7B). Together, these results show that EcfL and members of its regulon are required for full canker development in susceptible hosts.

FIG 7.

EcfL is required for full virulence of X. citri in citrus. (A) Virulence assay in Citrus sinensis using the WT, ΔecfL, Δ4130, and complemented X. citri strains. Complemented strains containing a wild-type copy of the deleted gene in the pBRA vector (ΔecfL/ecfL and Δ4130/4130) and strains carrying the empty vector were inoculated by the pinprick method (66, 72). Lesions around inoculation sites were photographed 20 days after infection. Results from measurements of a minimum of 30 inoculation sites for each strain are shown in the graph as relative lesion area, using data from the WT strain as a reference. Results are from a representative experiment of two biological replicates. (B) qRT-PCR analysis of expression of ecfL and EcfL target genes during growth of X. citri inside the plant leaf mesophyll. Samples obtained immediately after syringe injection (time = 0 h) were considered a reference for relative analysis. (C) ecfL and its target genes are induced by acidic pH in an EcfL-dependent manner. Mid-logarithmic-phase cultures of wild-type, ΔecfL, and Δ4130 strains were incubated for 30 min in medium pH 5.0 for qRT-PCR analysis. Samples harvested immediately before the pH shift were used as a reference for relative analysis. (D) qRT-PCR analysis of mRNA levels of the T3SS regulator hrpG and the T3SS gene hrcU in strains with altered levels of ecfL. The wild-type strain and the WT with empty vector were used as reference for relative quantification of mRNA levels in ΔecfL and WT/ecfL++ strains, respectively. qRT-PCR results in panels B, C, and D are the means of three independent experiments ± standard error bars. Results were normalized to rpoB as an internal control.

We tested the induction of ecfL and its regulon in response to acidic pH, a condition found in the plant leaf apoplast that induces T3SS secretion in X. campestris pv. vesicatoria and virulence gene expression in other nonvascular phytopathogenic bacteria (40–42). Results from qRT-PCR analysis showed that expression of ecfL and XAC4130 is increased 30 min after a shift to pH 5.0. In addition, XAC4131 and XAC4132 showed EcfL-dependent induction upon acidification of the culture medium (Fig. 7C). Therefore, acid pH is an environmental cue found in the plant environment that elicits the response mediated by EcfL. The Δ4130 strain showed similar induction levels of the target genes to those in the WT, indicating that the putative anti-σ does not affect EcfL activity under inducing conditions. We also tested whether EcfL is required for the response to iron starvation under acidic pH conditions. Deletion of ecfL did not cause increased sensitivity to iron starvation by addition of 2,2′-dipyridyl to LB or XVM2 adjusted to pH 5.0 (Fig. S7). In addition, iron starvation did not promote an exacerbated induction of the EcfL regulon in acidic pH conditions (Fig. S7C). These results further suggest that EcfL is not involved in the iron starvation response.

Results from transcriptome analysis under ecfL overexpression conditions in complex medium did not identify genes encoding known virulence factors as being regulated by EcfL (Fig. 2C). These experiments were performed using cultures grown in nutrient rich medium, a condition that typically suppresses expression of virulence genes in plant-pathogenic bacteria (41, 43, 44). Therefore, we decided to further investigate whether EcfL is involved in the regulation of the type III secretion system (T3SS)—the major virulence determinant in X. citri—during growth in XVM2 medium, a condition that induces expression of virulence factors, possibly by mimicking the plant environment (35, 45). We analyzed the mRNA levels of hrpG and hrcU, which encode a master regulator and a structural component of the T3SS (46, 47), respectively, in strains with altered levels of ecfL. Results from this analysis showed that hrpG and hrcU mRNA levels are not significantly influenced by deletion or overexpression of ecfL (Fig. 7D). These results suggest that EcfL does not promote virulence by regulating T3SS gene expression.

We also tested the role of EcfL and XAC4130 on the induction of hypersensitive response (HR) in nonhost plants, which is triggered by plant recognition of bacterial T3SS effectors upon infection (44). No difference was observed between the HR elicited by the WT and the mutant strains in bean (Phaseolus vulgaris) and tobacco plants (Nicotiana tabacum), as assessed by macroscopic symptoms and trypan blue staining of dead plant cells (Fig. S6B). These results further suggest that EcfL is not required for expression of the T3SS in planta.

DISCUSSION

FecI-like sigma factors form the largest group within the ECF family and are part of a cell surface signaling pathway (CSS) that involves an outer membrane TBDR with a sensor domain and an inner membrane regulator that transduces the signal from the outside of the cell to the cytoplasm. These regulators play fundamental roles in siderophore-mediated iron acquisition in several bacterial taxa and can control other aspects of bacterial physiology, including virulence gene expression, since iron starvation is a condition encountered in host tissues during infection (48). Here, we describe the functional characterization of a FecI-like ECF from X. citri, which is found in a restricted group of species within the Xanthomonas genus, as well as in a few other Xanthomonadaceae genera, including Stenotrophomonas. Distinct from most characterized members of the group, EcfL is not required for growth under iron starvation conditions. Moreover, ecfL and EcfL-regulated genes are not induced by this condition. Expression of FecI-like σ^ECF factors is often kept at low levels by the Fur transcriptional regulator during growth under iron-replete conditions, and induction occurs upon iron starvation (16). Consistent with our gene expression analysis, we did not identify a canonical Fur-binding box in the ecfL or XAC4131 promoters using the MEME motif search tool (https://meme-suite.org/meme/). Our results so far rule out a role for EcfL in siderophore acquisition in X. citri.

The phenotypic characterization of an ecfL mutant strain revealed that this σ factor is required for full virulence in citrus plants, since the ΔecfL strain caused milder disease symptoms, with some virulence still retained. In fact, no significant difference in disease progression was observed upon syringe inoculation of the ΔecfL strain with a higher inoculum dose (data not shown). These results indicate that EcfL is not essential for expression of virulence genes, but possibly affects the ability of X. citri bacteria to grow inside the plant mesophyll. In agreement with this hypothesis, results from RNA-Seq and qRT-PCR experiments showed that EcfL is not involved in regulation of the T3SS, the main virulence determinant of X. citri. In addition, induction of the hypersensitive response in nonhost plants (bean and tomato), which is also triggered by T3SS effectors, was not impaired by deletion of ecfL. Interestingly, Ralstonia solanacearum PrhI, the other FecI-like σ^ECF factor characterized in a plant pathogen, also participates in a cell signaling cascade involving the FecA-like TBDR PrhA that is not regulated by iron levels but is induced by plant cell contact and is required for full symptom development in tomato and Arabidopsis (14, 49). However, differently from EcfL, PrhI is encoded within the hrp T3SS-encoding gene cluster and is responsible for the induction of hrp genes, being also required for full HR response in this bacterium (14). Previous work has shown regulation of hrp genes by the XAC4131 homologue of X. citri strain NA-1 (50). However, our data do not support a similar model in X. citri strain 306.

Characterization of σ^ECF factors invariably involves the identification of their regulons, which provides essential information for further phenotypic studies. Transcriptome analysis of a strain overexpressing EcfL identified three induced genes (XAC4131-XAC4132-XAC4133) organized in a single transcriptional unit adjacent to the ecfL-XAC4130 genes, which is conserved among the closest EcfL homologues (Fig. 1B). Accordingly, the few Fec-like σ^ECF factors characterized to date typically control the expression of a few genes that are located in the vicinity of the σ^ECF-encoding gene and involved in a specific function, which is typically related to nutrient acquisition by a TBDR (4). XAC4131 encodes a TBDR with an amino-terminal extension found in members of the CSS (FecA-like), and its regulation by EcfL strongly suggests that the conserved signal transduction mechanism described in other bacteria controls the EcfL-mediated response. Importantly, whereas most bacterial genomes encode a small number of TBDRs (fewer than 14), bacteria from the Xanthomonas genus are part of a particular group that encodes more than 30 of these outer membrane transporters in their genomes (36). A functional study of the X. campestris pv. campestris TBDRs revealed that only 9 out of 72 are regulated by iron status, whereas 24 are predicted to be involved in the utilization of complex carbohydrates derived from plant cell walls (36). Therefore, the overrepresentation of TBDRs in Xanthomonas spp. suggests that they might be involved in the uptake of other plant-derived molecules, allowing bacterial growth in the phyllosphere, inside the mesophyll and in plant debris.

Comparative genome analysis allows for predictions of TBDR substrates based on patterns of colocalization and coregulation with genes encoding other nutrient transporters or hydrolytic enzymes (51). We demonstrate that XAC4131 is coregulated with XAC4132 and XAC4133 and that their genome context is conserved in the vicinity of other ecfL homologues, especially regarding co-occurrence with an alkaline/acid phosphatase-encoding gene (Fig. 1B). XAC4132 encodes a histidine acid phosphatase with homology to phytases, enzymes that catalyze the release of phosphate from phytate and confer the ability to use phytate as a phosphate source. Xanthomonas oryzae-secreted phytase PhyA is required for the use of phytate as the sole phosphate source, and the phyA mutant shows reduced virulence (52). Although we observed reduced virulence in the absence of the XAC4132 regulator EcfL, our data do not support an essential role for EcfL-regulated genes in the utilization of phytate as a sole phosphate source. Accordingly, the X. citri genome encodes a close homologue of X. oryzae PhyA, XAC2519 (91% identity), while XAC4132 does not present significant homology to PhyA (18% identity), suggesting that XAC4132 plays a distinct role in X. citri. The X. citri genome carries two XAC4132 paralogs, XAC0557 and XAC0812 (32% and 32.7% identity, respectively); the latter is also found adjacent to a TBDR gene. Interestingly, association with TBDR genes is a common feature among phytase homologues in microbial genomes, further suggesting a role in nutrient acquisition (53).

XAC4133, the third gene of the XAC4131-XAC4132-XAC4133 operon, encodes a conserved hypothetical protein of unknown function, with homologues found in Xanthomonas genomes that encode ecfL and in distantly related soil bacteria from Actinobacteria, including Streptomyces spp. XAC4133 carries a predicted signal peptide and a carbohydrate-binding lectin domain of the jacalin type, suggesting a role in recognition of cell surface-exposed glycan receptors, which might promote adhesion to host plant cells. Analysis of the XAC4133 sequence using the Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) identified a phosphatase domain of the exonuclease/endonuclease superfamily (EEP domain), the members of which act on diverse substrates, including phospholipids and nucleic acids. Based on the characteristics of proteins encoded by the EcfL regulon, results from our phenotypic characterization of the ΔecfL strain and the observed induction of ecfL and EcfL-regulated genes in the plant mesophyll and in response to acid pH conditions, we propose that EcfL is involved in the control of a nutrient acquisition pathway required for X. citri colonization of host plants, possibly involving phosphate-rich compounds. Studies are under way to better understand the role of XAC4132 and XAC4133 in X. citri.

XAC4130 encodes a predicted anti-σ factor with the conserved characteristics of the FecR group and we showed that its cytoplasmic amino-terminal domain interacts with EcfL, as shown for other members of this group. Besides their role as negative regulators of σ activity, several FecR-like proteins also have a positive effect on σ activity, by increasing affinity of the σ factor for the RNA polymerase core or stabilizing the σ factor, and in some cases, they are required for σ function, as shown for FecR, PupR, Pseudomonas aeruginosa FiuR, and R. solanacearum PrhR (16, 17). However, the positive effect on sigma factor activity is not a universal feature among these regulators, and some have been reported to act solely in negative regulation, including P. aeruginosa FpvR and Serratia marcescens HasS. Our results from characterization of the effects of altered cellular levels of XAC4130 on expression of the EcfL-regulated genes provided evidence for a positive effect of XAC4130 on EcfL activity, whereas a negative regulatory role was not evidenced by our analysis. Our data also suggest that XAC4130 is not required for EcfL function, since no defect in virulence was observed for the deletion strain. In addition, overexpression of EcfL was sufficient to induce its target genes. Further work is required to better characterize the mechanisms of regulation of EcfL activity by XAC4130 and a possible role in EcfL affinity for the RNA polymerase.

To the best of our knowledge, this work reports the first functional characterization of a member of the largest group of σ^ECF factors, ECF243, in Xanthomonas species, showing its role in virulence during infection of citrus plants. Work is under way in order to identify the molecular signal responsible for inducing EcfL expression and the possible role of the members of its regulon in nutrient scavenging by this bacterium. The X. citri genome encodes another member of this group (XAC2191), whose function remains elusive. Further studies to characterize ECF243 members in xanthomonads will broaden our understanding of these important regulators in this group of phytopathogens.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Strains used in this study are listed in Table S2 in the supplemental material. Xanthomonas citri strains were grown in LB broth (10 g · L⁻¹ peptone, 5 g · L⁻¹ yeast extract, and 10 g · L⁻¹ NaCl; pH 7), 2× TY broth (16 g · L⁻¹ tryptone, 10 g · L⁻¹ yeast extract, and 5 g · L⁻¹ NaCl) or XVM2 [20 mM NaCl, 10 mM (NH₄)₂SO₄, 5 mM MgSO₄, 1 mM CaCl₂, 0.16 mM KH₂PO₄, 0.32 mM K₂HPO₄, 0.01 mM FeSO₄, 10 mM fructose, 10 mM sucrose, and 0.03% Casamino Acids] at 28°C with shaking at 140 to 200 rpm. When required, the following antibiotics were added to X. citri cultures: 100 μg · mL⁻¹ ampicillin, 50 μg · mL⁻¹ kanamycin, and 50 μg · mL⁻¹ streptomycin. Escherichia coli strains were grown in LB broth with shaking at 140 to 200 rpm at 37°C. When required, 50 μg · mL⁻¹ kanamycin and 100 μg · mL⁻¹ streptomycin (final concentrations) were added to the medium.

Molecular cloning.

Plasmids, constructs and oligonucleotides used in this study are described in Tables S3 and S4 in the supplemental material. The in-frame deletion strains of ecfL (XAC4129 locus, ΔecfL) and XAC4130 (Δ4130) were generated by two-step allelic exchange (54). Fragments of approximately 1 kb flanking the regions to be deleted and including a few nucleotides from the 5′ and 3′ends of each coding sequence were amplified from genomic DNA by PCR. These fragments were joined in a second PCR step using external primers and by extension from the 12-nucleotide-long complementary region introduced in the internal primers in the first PCR step. The resulting fragments containing in-frame deletions of most of the ecfL and XAC4130 coding regions (deletions of 396 bp and 959 bp, respectively) were cloned into pNPTS138 suicide vector using HindIII and EcoRI restriction enzymes (Thermo Scientific). The constructs were introduced into X. citri by electroporation and integrated into chromosome by two-step homologous recombination. For the first recombination event, transformants were selected based on their resistance to kanamycin and sensitivity to 5% sucrose in LB agar. For the second recombination step that eliminates the vector with one copy of the gene (WT or deleted version), individual colonies were grown in LB medium without antibiotic and spread in LB agar with 5% sucrose. Individual colonies were further screened for kanamycin sensitivity. Mutant strains containing only the deleted version were confirmed by PCR. Constructs for overexpression and complementation of ecfL and XAC4130 were obtained by PCR amplification from genomic DNA and subsequent cloning in the pBRA vector, a derivative of pBAD24 (M. Marroquim, unpublished data). Low constitutive expression of the cloned gene is observed in the absence of arabinose, and expression is repressed by addition of glucose (data not shown). Plasmids were transformed into X. citri by electroporation, and selection was performed in streptomycin plates. All constructs obtained by PCR amplification were verified by DNA sequencing to confirm sequence integrity.

RNA-Seq sample extraction, cDNA library preparation, and sequencing.

X. citri cultures were diluted to an optical density at 600 nm (OD₆₀₀) of 0.1 in 2× TY broth, then incubated at 28°C with shaking at 140 to 200 rpm until they reached an OD₆₀₀ of 0.5 to 0.6. Cells (6-mL cultures) were harvested by centrifugation at 16,000 × g for 1 min and lysed immediately by homogenization with 1 mL of TRIzol reagent (Ambion). Samples were incubated at 65°C for 10 min, followed by addition of 0.2 mL of chloroform per 1 mL of TRIzol used. Samples were incubated for 5 min at room temperature and centrifuged for 15 min at 20,000 × g (4°C). The aqueous phase was harvested, and 0.5 volume of cold ethanol 100% was added. This last step of RNA purification was performed using filter cartridges and wash and elution solutions from the RiboPure bacteria kit (Ambion/Life Technologies), following the manufacturer’s instructions. Total RNA was treated with Turbo DNA-free DNase (Ambion/Life Technologies) according to the manufacturer’s recommendation. Sample quality was confirmed using a 2100 Bioanalyzer (Agilent Technologies). Removal of rRNA was performed using 5 μg of each sample and the Ribo-Zero Gram-negative bacteria rRNA removal kit (Illumina). Libraries were prepared with TruSeq RNA library preparation LT v2 (Illumina) and quantified using a library quantification kit for Illumina platforms (KAPA Biosystems). The libraries were normalized to 4 nM and sequenced by NextSeq Illumina platform at the CEFAP/Genial core facility “Core Facility for Scientific Research—University of Sao Paulo (USP) (CEFAP-USP)” using a NextSeq 500/550 mid-output v2 kit (150 cycles, 2 × 75).

RNA-Seq differential expression analysis.

RNA-Seq data were processed using the frtc pipeline (https://github.com/alanlorenzetti/frtc/) (55). In summary, we inspected raw data for quality using Rqc (56) and trimmed reads to remove sequencing adapters and low-quality ends using Trimmomatic (57). We aligned reads to Xanthomonas citri RefSeq genome ASM716v1 (NCBI assembly accession number GCF_000007165.1) using HISAT2 set to prevent spliced alignments (58). We processed uniquely aligned reads using SAMtools (59) and computed the count matrix using GenomicAlignments (60). We performed differential expression analysis using DESeq2 (61) and detected significant changes by selecting genes satisfying a log₂ fold change of more than 1 or less than −1 and an adjusted P value of <0.01. We also processed read alignments using deepTools2 to create single-nucleotide resolution read depth files (62), which were inputted in Integrative Genomics Viewer for genome-wide visualization (63).

RNA extraction and qRT-PCR.

Samples for qRT-PCR analysis of gene expression under standard growth conditions and in response to acid pH (LB, pH 5.0) were obtained from exponential-phase cultures grown in LB, 2× TY or XVM2, as indicated for each experiment. For analysis of mRNA levels in response to acid pH, bacteria were cultivated in LB (pH 7.0) until the exponential phase, washed three times with water, and resuspended in LB (pH 5.0). Samples for gene expression analysis during infection of host plants were obtained as previously described (64). Briefly, saturated cultures of X. citri pv. citri were washed and resuspended in 10 mM MgCl₂ and inoculated by syringe infiltration into the abaxial surface of sweet orange leaves [Citrus sinensis (L.) Osbeck]. Plants were kept under greenhouse conditions (18-h photoperiod), and six leaves were removed from two plants (three leaves from each plant) at the time points of interest and combined into one sample. Leaves were ground using a mortar and pestle. Bacterial cells were harvested by centrifugation and mixed with TRIzol reagent for extraction of total mRNA.

For mRNA extraction, samples (2 mL) were harvested by centrifugation and resuspended in 1 mL TRIzol reagent (Ambion), and total RNA purification was performed by chloroform extraction followed by isopropanol precipitation, following the manufacturer’s instructions. Total mRNA was treated with DNase I (Thermo Scientific) and absence of DNA contamination was checked by PCR. Synthesis of cDNA was performed using a RevertAid H Minus first-strand cDNA synthesis kit (Thermo Scientific) using 500 ng of mRNA. Quantitative real-time PCRs were performed with 50 ng of cDNA as the template, 0.4 mM primers (Table S4), and SYBR green qPCR master mix (Thermo Scientific). Relative mRNA levels were determined using the 2^−ΔΔCT method, with samples normalized to rpoB or rpoC genes (65).

5′ RACE.

Total RNA was obtained as described above and used as a template for determination of the XAC4131 transcription start site using the 5′/3′ RACE kit (2nd generation; Roche/Sigma-Aldrich) as recommended by the supplier. In brief, exponential-phase cultures of the WT/ecfL⁺⁺ strain were harvested by centrifugation for mRNA extraction with TRIzol reagent. mRNA samples were treated with DNase, reverse transcription was performed with the antisense-specific primer xac4131-SP1 (Table S4), and the poly(A) tail was added to the resulting cDNA. The poly(A)-tailed cDNA was amplified by PCR using the oligo(dT) anchor primer provided by the kit and the antisense gene-specific primer xac4131-SP2 (Table S4), with Phusion high-fidelity DNA polymerase (Thermo Scientific). A second PCR was performed using the gene-specific primer xac4130-F2rev (Table S4) and PCR anchor primer provided in the kit. The agarose gel band corresponding to the amplification product was extracted and purified using the GeneJet gel extraction kit (Thermo Scientific). The purified product was cloned in the pGEM-T vector (Promega). Clones obtained were sequenced by the Sanger method at the GENIAL/CEFAP facility of the University of São Paulo (ICB/USP).

Virulence and HR assay.

Virulence assays were performed with the sweet orange variety ‘Natal’ (Citrus sinensis), which was maintained in greenhouse conditions. Bacterial strains were cultivated for 48 h in LB agar and resuspended in water to an OD₆₀₀ of 0.1. Bacterial suspensions were inoculated using pin pricks, as described previously (66). Each leaf was inoculated in 16 spots, and a minimum of three leaves were used for individual bacterial strains in each experiment. Inoculated plants were monitored daily for symptom development. Lesion areas were determined 20 days after inoculation using ImageJ software, and statistical analysis was performed by paired-sample t test using Origin 8.1.

Growth curves.

Overnight cultures in LB were washed and diluted to an OD₆₀₀ of 0.1 under the growth condition of interest. Growth curves of strains carrying pBAD constructs for overexpression of σ factors were performed in 2× TY broth medium supplemented with 0.3% glucose or 0.3% arabinose. Bacterial viability was determined from cultures cultivated as described above by serial dilution and plating in LB supplemented with 0.3% glucose. For iron starvation experiments, cultures were diluted in XVM2 containing 200 μM 2,2′-dipyridyl (an iron chelator), and control conditions consisted of XVM2 without the chelator. For phosphate starvation growth curves, cultures were diluted in modified Miller’s M4 minimal medium (52, 67) (control condition, phosphate-replete medium), low-phosphate M4 medium (LPM4; M4 containing 2 mM K₂HPO₄; 1 mM KH₂PO₄), or LPM4 supplemented with 3 mM phytic acid.

Phagocytosis plaque assay.

Experiments were performed as described previously (68). Overnight bacterial cultures were washed in LB medium, normalized to an OD₆₀₀ of 3.0, and diluted 100× in LB. Diluted cultures (50 μL) were spread in individual wells of 24-well plates containing standard medium (SM) (bacteriological peptone 10 g/L, yeast extract 1 g/L, KH₂PO₄ 2.2 g/L, K₂HPO₄ 1 g/L, MgSO₄ · 7H₂O 1 g/L, 20 g/L agar, and glucose 10 g/L [pH 6.5]) and allowed to dry for 60 min in a laminar flow hood. D. discoideum cells were washed in development buffer (DB; 5 mM KH₂PO₄, 5 mM Na₂HPO₄, 1 mM CaCl₂, and 2 mM MgCl₂ [pH 6.5]), counted, and cell concentration was adjusted in order to inoculate in each well the desired number of cells in spots of 5 μL. Plates were incubated for 3 to 5 days at 22°C, and formation of phagocytic halos was observed throughout the incubation period.

Yeast two-hybrid assay.

Constructs encoding protein fusions of XAC4130_1–117 or EcfL to the activation or the DNA binding domain of the transcription factor Gal4 were obtained by PCR amplification from X. citri genomic DNA and subsequent cloning in the pOAD (XAC4130_1–117) and pOBD vectors (ecfL). Primers used for amplification are described in Table S2. Assays were performed after cotransformation of Saccharomyces cerevisiae strain PJ69-4a (69) with the pOAD and pOBD constructs, as previously described (70). For protein interaction tests, S. cerevisiae strains were cultivated in SC solid medium (0.66% nitrogen base [without amino acids], 2% glucose, 0.008% adenine, 0.8% amino acid mixture, 3 mM 3-aminotriazole, and1.6% agar [pH 5.6]) lacking one or more of the following amino acids: histidine (−His), leucine (−Leu), tryptophan (−Trp), and/or adenine (−Ade).

Data availability.

Raw RNA-Seq data are available at the Sequence Read Archive under BioProject accession number PRJNA736807.