Advertisement

BMC Genomics

, 20:55 | Cite as

Diffusible signal factor (DSF)-mediated quorum sensing modulates expression of diverse traits in Xanthomonas citri and responses of citrus plants to promote disease

  • Lei Li
  • Jinyun Li
  • Yunzeng Zhang
  • Nian WangEmail author
Open Access
Research article
Part of the following topical collections:
  1. Prokaryote microbial genomics

Abstract

Background

The gram-negative Xanthomonas genus contains a large group of economically important plant pathogens, which cause severe diseases on many crops worldwide. The diffusible signal factor (DSF) - mediated quorum sensing (QS) system coordinates expression of virulence factors in plant pathogenic Xanthomonas spp. However, the regulatory effects of this system during the Xanthomonas- plant interactions remain unclear from both the pathogen and host aspects.

Results

In this study, we investigated the in planta DSF- mediated QS regulon of X. citri subsp. citri (Xac), the causal agent of citrus canker. We also characterized the transcriptional responses of citrus plants to DSF-mediated Xac infection via comparing the gene expression patterns of citrus trigged by wild type Xac strain 306 with those trigged by its DSF- deficient (∆rpfF) mutant using the dual RNA-seq approach. Comparative global transcript profiles of Xac strain 306 and the ∆rpfF mutant during host infection revealed that DSF- mediated QS specifically modulates bacterial adaptation, nutrition uptake and metabolisms, stress tolerance, virulence, and signal transduction to favor host infection. The transcriptional responses of citrus to DSF-mediated Xac infection are characterized by downregulation of photosynthesis genes and plant defense related genes, suggesting photosynthetically inactive reactions and repression of defense responses. Alterations of phytohormone metabolism and signaling pathways were also triggered by DSF-mediated Xac infection to benefit the pathogen.

Conclusions

Collectively, our findings provide new insight into the DSF- mediated QS regulation during plant-pathogen interactions and advance the understanding of traits used by Xanthomonas to promote infection on host plants.

Keywords

Xanthomonas Diffusible signal factor (DSF) Quorum sensing Citrus canker Transcriptomic profiling 

Abbreviations

bp

Base pair

cDNA

Complementary DNA

COG

Clusters of Orthologous Groups

DEGs

Differentially expressed genes

DNA

Deoxyribonucleic acid

DSF

Diffusible signal factor

EPS

Extracellular polysaccharides

FDR

False rate discovery

FPKM

Fragments per kilobase of exon permillion mapped reads

kb

Kilobases

log2FC

Log of fold change in base 2

LPS

Lipopolysaccharides

min

Minute

mM

Millimolar

mRNA

Messenger RNA

PCR

Polymerase chain reaction

pH

Hydrogenionic potential

qRT-PCR

Quantitative reverse transcription PCR

QS

Quorum sensing

RNA

Ribonucleic acid

RNA-Seq

RNA sequencing

Rpf

Regulation of pathogenicity factorsL

rpm

Rotations per minute

T2SS

Type II secretion system

T3SS

Type III secretion system

Xac

X. citri subsp. citri

Xcc

X. campestris pv. campestris

μg

Micrograms

μM

Micromolar

Background

The genus Xanthomonas comprises a large group of gram-negative plant pathogenic bacteria that have considerable agricultural impact worldwide, and therefore, is an important model genus for studying the host-pathogen interactions [1, 2]. Successful infection and bacterial multiplication of Xanthomonas spp. in host tissues require coordinated expression of a combination of virulence factors. Key virulence factors of Xanthomonas spp. include, among others, the type III secretion system (T3SS) and its effectors [3, 4], bacterial polysaccharides such as the xanthan extracellular polysaccharides (EPS) and lipopolysaccharides (LPS) [5], and cell wall degrading enzymes [1]. Expression of these virulence factors is regulated by different extracellular stimuli via multiple coordinated regulatory systems, including cell-to-cell communication (quorum-sensing, QS) pathways, two-component systems and various transcriptional regulators [1].

The QS regulatory systems of Xanthomonas are mediated by molecules belonging to the diffusible signal factor (DSF) family [2, 6, 7]. The DSF-mediated QS has been studied most extensively in the crucifer pathogen X. campestris pv. campestris (Xcc). The synthesis and perception of the DSF signal, which was identified as cis-11-methyl-2-dodecenoic acid [8], require the rpf gene cluster (for regulation of pathogenicity factors), including rpfB, rpfF and rpfGHC [9, 10]. RpfB was initially thought to be involved in DSF biosynthesis, but it was later identified as a fatty Acyl-CoA ligase involved in the turnover of the DSF family of signals in Xanthomonas [11]. The RpfF protein, functioning as a putative enoyl-CoA hydratase, is responsible for the synthesis of DSF. RpfC and RpfG consist of a two-component system involved in DSF perception and signal transduction. RpfC is a hybrid sensor kinase and RpfG is a response regulator with a CheY-like receiver (REC) domain and an HD-GYP domain, capable of degrading the second messenger cyclic di-GMP [6, 10, 12, 13]. DSF can bind directly to the N-terminal, 22 amino acid-length sensor region of RpfC and activate RpfC autokinase activity to regulate QS and virulence in Xcc [14]. RpfH is a putative membrane protein with no known role in DSF signaling [10].

The contribution of DSF/Rpf regulatory system to virulence has been demonstrated in many members of Xanthomonas. For example, DSF signaling in Xcc influences the synthesis of a range of virulence factors including extracellular enzymes such as endoglucanase, protease, and endomannanase, and the xanthan EPS, as well as alterations in biofilm formation [6, 10, 15]. Specifically, the RpfS- dependent second DSF signaling pathway controls expression of genes involved in type IV secretion and chemotaxis and therefore affects bacterial motility, suggesting a role in the epiphytic phase of the Xcc disease cycle [16]. Similarly, the DSF-mediated QS has been shown involved in early attachment and in planta growth of Xac in the citrus host during the citrus canker disease cycle [17]. Recent report indicates that the DSF family in Xcc elicited plant innate immunity and this effect was suppressed through the secretion of the xanthan exopolysaccharide [18]. DSF also confers a fitness advantage to Xcc during interspecies competition [19].

Transcriptome profile, functional genomics, and proteomic analyses have significantly advanced the understanding of the DSF/Rpf regulatory network and its role in pathogenesis of Xanthomonas. Earlier studies have revealed that the RpfC/RpfG two-component system coordinately regulates the expression of various genes related to virulence via the cyclic di-GMP signaling that activates the transcriptional activators Clp and Zur in Xcc [6, 12, 13, 20]. These include the genes encoding extracellular enzymes, components of type II secretion system (T2SS), components of type III secretion system (T3SS), and the genes involved in EPS production. Comparative transcriptome studies using whole-genome microarray showed that the DSF/Rpf -mediated QS regulation in the citrus canker pathogen X. citri subsp. citri (Xac) is growth phase-dependent, and more genes in the exponential phase are differentially regulated by the RpfC/RpfG system compared with in the stationary phase [17]. The RpfC/RpfG system-regulated genes include diverse genes involved in chemotaxis and motility, flagellar biosynthesis, production of extracellular enzymes and adhesins, stress tolerance, regulation, transport, and detoxification [17]. There are also some unique genes controlled by RpfF, RpfC or RpfG alone, indicating the complexity of the QS pathway and the involvement of additional DSF signal perception and transduction mechanisms in Xac [17]. Interestingly, recent studies suggested additional signaling outputs from RpfC and an interaction of RpfG with a second unknown sensor [16, 21]. The authors found that DSF and RpfC also regulate expression of a number of genes encoding transcriptional regulator, hydrolase, protease and hypothetical proteins independently of RpfG, and RpfG regulates expression of genes involved in chemotaxis, signal transduction and protein export, independently of RpfF or RpfC [16, 21]. These studies also revealed that RpfC can recognize other unidentified environmental signals (in addition to DSF) [21] and the DSF signal can be recognized by a second sensor RpfS, a PAS domain-containing histidine kinase that regulates genes involved in type IV secretion and chemotaxis in a pathway independent of RpfC and RpfG [16]. Our knowledge of the protein(s)/regulator(s) acting downstream of RpfS in DSF signal transduction cascades remains limited. In addition, the DSF/Rpf system controls three non-coding RNA (ncRNAs) that contribute to virulence in Xcc [21].

Comparative proteomic analysis revealed diverse regulatory effects of DSF/Rpf in Xcc on proteins involved in regulation, biosynthesis and intermediary metabolism, stress tolerance, and motility [22]. Similarly, mutation of the rpfF gene has a substantial impact on the proteome of X. oryzae pv. oryzicola, affecting proteins involved in a range of functions including nitrogen transfer, protein folding, resistance to oxidative stress and flagellar synthesis [23]. Interestingly, for many of the proteins regulated by the DSF/Rpf system in Xcc, the alteration in abundance was not associated with alteration in transcript level, suggesting that both posttranscriptional regulation and post-translational turnover may occur [22].

Despite the extensive transcriptome analyses of the DSF/Rpf regulatory system in Xanthomonas spp. as stated above, most of which were performed using the bacterium grown under culture media conditions, and knowledge on regulatory effects of the DSF/Rpf system of Xanthomonas spp. during the interaction with host plants is still lacking. The actions of the elements involved in DSF signaling and the role of DSF signal transduction during plant infection remain to be determined from both the pathogen and host aspects. In the present study, we investigated the DSF/Rpf QS regulation in planta during Xac infection of citrus host. Metatranscriptome analysis of the compatible interaction between Xac and citrus was conducted using RNA-Seq to compare the global transcriptomes of wild-type and isogenic rpfF mutant (∆rpfF) strains of Xac, as well as the citrus transcriptional patterns in response to their infection. This work provides a comprehensive picture of the genes and traits regulated by the DSF/Rpf QS in Xac in planta and host responses to the DSF-mediated infection.

Methods

Bacterial strains and growth conditions

Xanthomonas citri subsp. citri (Xac) wild type strain 306 [24] and its rpfF gene deletion (∆rpfF) mutant strain [17] were grown at 28 °C with shaking at 200 rpm. in nutrient broth (NB; Difco, Detroit, MI) containing rifamycin (50 μg/mL). Bacterial growth was measured in a spectrophotometer at 600 nm.

Plant inoculations and sampling of infected leaves

Plant inoculation was performed as described in our previous work [5]. Briefly, young (about 12-week-old) Duncan grapefruit (Citrus paradise Macfadyen) plants were grown in potting medium/soil in a greenhouse at the Citrus Research and Education Center, Lake Alfred, FL, USA, and maintained at approximately 25–30 °C and a 55% relative humidity until the primary leaves were fully expanded but not fully matured. The bacterial inoculum cells were grown as described above. When the cells reached late-log phase (OD600 = 1.0; 5 × 1010 cfu/mL), they were collected by centrifugation at 4000 rpm for 15 min. The cell pellets were resuspended in sterilized 0.85% NaCl, washed, and resuspended in sterilized 0.85% NaCl to a final density of 5 × 106 cfu/mL. To establish in planta populations, bacteria were introduced by infiltration into leaves using a needleless syringe. Infiltrated plants were maintained in the same greenhouse for canker symptom development. All plant inoculations included at least three leaves at a similar developmental stage from each plant, and ten replicate plants were inoculated for each strain. Time- course bacterial growth in planta was tested as described previously [5]. All the tests were repeated three times. Based on the progression of development of canker symptoms, the infected leaves were sampled at 5 days post inoculation (DPI) for RNA extraction. The inoculated leaf area was collected using a cork borer (leaf area, 1 cm2) and 10 leaf samples from each biological replicate (three replicates for each treatment) were pooled and immediately frozen in liquid nitrogen, and kept in − 80 °C until process for RNA isolation.

RNA extraction, library construction and Illumina RNA-seq

Total RNA was extracted from leaf samples using RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and contaminated DNA was removed by treatment with RNase-Free DNase Set (Qiagen, Valencia, CA). The quality and quantity of RNA samples were assessed using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and agarose gel electrophoresis. The total RNA was treated with DNase I (New England Biolabs, Ipswich, MA) prior to library construction. The rRNA of plant and bacteria was depleted using Ribo-Zero™ rRNA Removal Kits (Plant Leaf) and Ribo-Zero™ rRNA Removal Kits (Bacteria) respectively, according to the manufacturer’s instructions (Epicenter Technologies, Madison, WI). Poly (A) + mRNA was purified using Agencourt RNAClean XP Kit (Beckman Coulter Life Sciences, Indianapolis, IN) and fragmented into short pieces. Using these short fragments as templates, first strand cDNA synthesis was conducted using random hexamer-primers and SuperScript® II Reverse Transcriptase (Invitrogen, Waltham, MA), and the second-strand cDNA was synthesized using RNase H (Invitrogen, Waltham, MA) and DNA polymerase I (New England Biolabs, Ipswich, MA). After purification, end repair, and ploy (A) tails add, the cDNA fragments were ligated to sequencing adapters. Then fragments of an appropriate size were purified and amplified by PCR to produce the final library. Finally, the cDNA libraries were loaded onto the flow cell channels of an Illumina HiSeqTM 2000 platform for paired-end 90 bp × 2 sequencing at the Beijing Genomics Institute (BGI), Hongkong, China. Clean reads were obtained after removing reads containing adaptor sequences. The RNA reads have been deposited at NCBI under the bioproject No. PRJNA421992 with the SRA accession no. SRP126698.

Reads mapping and differential expression analysis

The clean reads were firstly aligned to the Xac strain 306 genome (https://www.ncbi.nlm.nih.gov/nuccore/AE008923.1/) [24] using bowtie2 [25] with default parameters. The in planta differential expressed genes between wild type Xac 306 and ∆rpfF mutant strains were identified using DESeq2 R package [26] with the following cutoffs: |fold change| ≥ 2 and agjust-P ≤ 0.05. After aligned to Xac strain 306 genome, the remaining reads from each sample were analyzed mainly following the tuxedo pipeline [27]. Briefly, the reads were aligned to the sweet orange genome [28] using Tophat (v2.0.13) [29], and the generated alignments were fed to Cufflinks (v2.2.1) for transcript assembly [30]. The assemblies were combined with the sweet orange annotations using the cuffmerge algorithm and then fed to the cuffdiff2 for differentially expressed gene calling. Only the genes with |fold change| ≥ 2, q-value ≤0.05 and FPKM≥1 were considered as significantly differentially expressed genes (DEGs) between wild type strain infected and ∆rpf mutant strain infected plants. The MapMan gene functional categories were assigned to the DEGs using Mercator [30, 31] and the differentially regulated bins were identified by using MapMan [32].

Functional annotation and classification

For citrus DEGs, the corresponding reference ID were obtained by blasting them to CitrusPLEX in plant expression database (PLEXdb, http://www.plexdb.org/plex.php?database=Citrus) [33]. Gene Ontology (GO) enrichment analysis of functional significance was applied to map all DEGs to terms in the agriGO (http://bioinfo.cau.edu.cn/agriGO/) database [34], looking for significantly enriched GO terms in DEGs. For bacterial DEGs, Clusters of Orthologous Groups (COG, https://www.ncbi.nlm.nih.gov/COG/) enrichment analysis was performed by comparing the prevalence of DEGs assigned to a specific COG category to the prevalence of genes in the whole genome assigned to that COG category with a Fisher’s exact test.

Validation of RNA-seq results by qRT-PCR

To verify the RNA-seq result, qRT-PCR assays were conducted using the same set of RNA samples as for RNA-seq analysis. The aliquoted RNA sample (1 μg) used for RNA-seq was reverse transcribed using a QuantiTect Reverse Transcription kit with random hexamer primers (Qiagen, Valencia, CA) for two-step qRT-PCR. The gene specific primers (Additional file 1: Table S1) were designed to generate amplicons of 70 to 150 bp based on the DEGs sequences of citrus plant and Xac strain 306. qRT-PCR was conducted using QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA) and the 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA). Melting curve analysis of the PCR products was performed at the end of each PCR cycle to confirm the amplicon specificity. The housekeeping gene CtGAPDH [35] and gyrA [5] was used as plant and bacterial endogenous control, respectively. All reactions were repeated with three independent biological replicates and two technical replicates. The relative fold change in target gene expression was calculated by using the formula 2-△△CT [36].

Statistical analysis

Quantitative data were expressed as mean ± S.E.M. Statistical differences were evaluated through t-test and the level of statistically significant difference was set at P<0.05. All statistics were conducted using SAS 9.1.3.

Results

Canker progression and symptoms in inoculated citrus plants

Duncan grapefruit (Citrus paradisi Macfadyen) seedlings were inoculated with Xac wild type strain 306 and its DSF deficient (∆rpfF) mutant strain for the development of typical symptoms of citrus canker. The first visible symptom, a water soaking area of the inoculated leaf, was observed at 5 days post inoculation (DPI). Within 14 DPI, typical symptoms of the canker disease were recorded (Fig. 1a): inoculated areas were characterized with water soaking, and then exhibited hyperplasia and hypertrophic, necrotic, erumpent lesions, as evidenced by the raised pustules. The ∆rpfF mutant produced weaker water soaking phenotypes compared to wild type strain 306 at 5 DPI under the tested conditions; and this becomes more evident at 7DPI and until 9 DPI (Fig.1a). However, the bacterial populations of the ∆rpfF mutant in planta were not significantly lower than the wild type strain (Fig. 1b).
Fig. 1

Citrus canker disease development in Xanthomonas citri inoculated Duncan grapefruit plants. a Representative leaves from ten replicates to show the development of canker symptoms on leaves inoculated with Xanthomonas citri subsp. citri wild type (Wt) strain 306 and its ∆rpfF mutant (M) with bacterial solutions (5 × 106 CFU/ml) by infiltration using needleless syringes and photographed at different days post inoculation (DPI). b Bacterial population growth in in Duncan grapefruit leaves inoculated with bacteria (5 × 106 CFU/ml) at different days post inoculation. Error bars represent standard deviation. All the experiments were repeated three times

Sequencing the early citrus canker transcriptome

As the differences in the development of canker symptoms only were recorded in early stages of disease development (formation of water-soaking phenotypes) between the wild type Xac and ∆rpfF mutant (Fig. 1a), we speculated that the DSF/Rpf QS play certain role(s) in early stages of the Xac-citrus compatible interaction. Therefore, the early canker transcriptome during the formation of water-soaking phenotypes was profiled at 5 DPI.A total of 227 million and 278 million paired-end reads for wild type Xac strain 306 infected and the ∆rpfF mutant infected plants were produced respectively (Additional file 2: Table S2). All reads were aligned against the Xac strain 306 genome [24]. For each RNA-seq library, 2.4–5.5% of the reads mapped to the Xac 306 reference. Then the remaining Xac strain 306-unaligned reads were mapped against the sweet orange (Citrus sinensis) genome [28], for the analysis of the citrus host transcriptome. A significant fraction of the Xac306-unaligned reads (63–68%) from both wild type Xac infected and ∆rpfF mutant infected libraries mapped to the sweet orange reference (Additional file 2: Table S2). Finally, of the 4374 Xac genes, 202 (4.5%) were determined as significantly differentially expressed genes (DEGs) [a minimum absolute value of a log2-fold change greater than 1 (equivalent to two-fold)] between wild type Xac 306 and ∆rpfF mutant strains in the conditions analyzed (Additional file 3: Table S3). Among them, 138 were upregulated in Xac wild type strain 306 compared to ∆rpfF mutant and 64 were downregulated. Of the 29,445 citrus (sweet orange) genes, 1946 genes were identified as significantly DEGs between wild type Xac 306 infected and ∆rpfF mutant strain infected plants, with 708 genes upregulated and 1238 downregulated in the wild type Xac 306 infected plants compared to ∆rpfF mutant strain infected plants (Additional file 4: Table S4).

To validate the gene expression values obtained by RNA-seq, the expression of 40 Xac genes and 33 citrus genes (Additional file 1: Table S1) in the RNA samples used in RNA-seq analysis were analyzed by qRT-PCR assays. A strong correlation (R2 = 0.9141 for Xac gene expression; R2 = 0.9011 for citrus gene expression) were observed between the data produced by the two approaches (Fig. 2a-b), demonstrating the reliability of the results obtained.
Fig. 2

Correlation analysis of expression levels of selected bacterial genes and citrus genes determined by RNA-seq and RT-qPCR. a Comparison of RNA-seq and qRT-PCR data for differentially expressed genes (DEGs) in Xanthomonas citri subsp. citri. Fold changes were calculated for 40 bacterial genes and a high correlation (R2 > 0.90) was observed between the results obtained using the two techniques. b Comparison of RNA-seq and qRT-PCR data for DEGs in citrus. Fold changes were calculated for 33 citrus genes and a high correlation (R2 > 0.90) was observed between the results obtained using the two techniques

Functionally categorizing of Xac genes regulated by the DSF/Rpf-mediated QS system during early stages of host infection

The 202 DEGs of Xac were subject to functionally categorizing with enrichment analyses of clusters of orthologous groups (COGs). The results showed that overrepresented COGs terms were mostly related to ‘Carbohydrate transport and metabolism’ (39 members, 16.3%), ‘Amino acid transport and metabolism’ (29 members, 14.4%), ‘Inorganic ion transport and metabolism’ (21 members, 10.4%), and ‘Cell wall/membrane/envelope biogenesis’ (19 members, 9.41%) (Fig. 3). Other enriched terms included ‘Lipid transport and metabolism’, ‘Energy production and conversion’, ‘Post-translational modification, protein turn over, and chaperones’, ‘signal transduction mechanisms’, ‘Transcription’, and ‘General function prediction only’. In addition, the genes annotated as hypothetical proteins were assigned to the ‘Function unknown’ group.
Fig. 3

Distribution of differentially expressed genes (DEGs) of Xanthomonas citri subsp. citri in COG functional categories. The x-axis represents the relative abundance (%) of DEGs and all the genes in the bacterial whole genome in each COG category. The y-axis represents the functional classification each COG category

DSF/Rpf-mediated QS regulates stress tolerance of Xac during early stages of host infection

A total 12 genes encoding enzymes involved in detoxification and stress tolerance of Xac at early stages of host infection were differentially regulated by DSF/Rpf-mediated QS (Table 1). Of these, the genes coding for a putative arabinose efflux permease belonging to the Major Facilitator Superfamily (MFS) transporter for sugar/drug (araJ /XAC1363), for a drug resistance translocase (yieO /XAC2494), for an endoproteinase (argC/XAC2992), and for trehalose biosynthesis (XAC0425 and XAC0429) were upregulated by ≥2-fold on average. Bacterial endoproteinases are able to degrade host defense proteins [37, 38], and trehalose protects bacterial cells from osmotic and oxidative stresses [39, 40]. The katE gene (XAC1211) encoding a catalase important for hydrogen peroxide torelance in Xac [41], was also upregulated by 2-fold.
Table 1

List of genes related to stress tolerance in Xac regulated by DSF/Rpf-mediated QS during early stages of host infection

Locus tag

Gene name

Log2Fold Change (Wt/∆rpfF)

Annotation/ Protein function

XAC0425

glgA

1.03

glycogen synthase (trehalose biosynthesis)

XAC0429

glgY

1.04

malto-oligosyltrehalose synthase

XAC1211

katE

1.00

catalase

XAC1363

araJ

1.32

arabinose efflux permease, MFS transporter

XAC1927

aslB

1.14

Fe-S oxidoreductase, stress-responsive

XAC2494

yieO

1.29

drug resistance translocase

XAC2992

argC

2.98

endoproteinase Arg-C, degrading host defense proteins

XAC4259

blc

1.05

lipocalin, involved in detoxification processes

XAC0906

ahpF

−1.01

alkyl hydroperoxide reductase scavenging H2O2

XAC0907

ahpC

−1.14

alkyl hydroperoxide reductase scavenging H2O2

XAC3486

fabG

−3.14

3-ketoacyl-ACP reductase, induced by nutrient limit conditions

XAC4361

ttuB

−1.51

MFS transporter

DSF/Rpf -mediated QS is implicated in the regulation of nutrition utilization of Xac during early stages of host infection

A significant portion of the Xac in planta transcriptome regulated by DSF/Rpf -mediated QS is dedicated to nutrition utilization (Fig. 3). Of the 39 genes involved in carbohydrate uptake and metabolism, 14 were positively regulated by DSF/Rpf -mediated QS, while 25 were negatively regulated (Table 2). The carbohydrate genes upregulated by DSF/Rpf –mediated QS included those encoding cellulose endoglucanase (egl/XAC0029 and engXCA/XAC0612), glycosyl transferase (gtrB/XAC1038/XAC2125, XAC3533, and ugt/XAC3921), glycosyl hydrolase (XAC3073), glycogen synthase (glgA /XAC0425 and glgY/XAC0429), glucose dehydrogenase (gcd/XAC1633/XAC3212), glucokinase (glk/XAC3120), and transporter (araJ/XAC1363 and yieO/XAC2494). In contrast, the expressions of fruBK and fruA encoding components of a fructose-specific phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) were downregulated.
Table 2

List of genes involved in nutrient transport or metabolism in Xac regulated by DSF/Rpf-mediated QS during pathogenic process

Locus tag

Gene name

Log2Fold Change (Wt/∆rpfF)

Annotation/ Protein function

Carbohydrates transport and metabolism

XAC0029

egl

1.34

cellulase

XAC0425

glgA

1.03

glycogen synthase

XAC0429

glgY

1.04

malto-oligosyltrehalose synthase

XAC0612

engXCA

1.53

cellulase

XAC1038

gtrB

1.12

glycosyl transferase

XAC1363

araJ

1.32

MFS transporter

XAC1633

gcd

2.06

glucose dehydrogenase

XAC2125

gtrB

1.07

glycosyl transferase

XAC2494

yieO

1.29

drug resistance translocase

XAC3073

 

1.00

GH18 family; chitinase-like glycosyl hydrolase

XAC3120

glk

1.36

glucokinase

XAC3212

gcd

1.05

glucose dehydrogenase

XAC3533

 

1.23

Glycosyltransferase, GT2 family

XAC3921

ugt

1.52

glucosyltransferase

XAC0217

lgtB

−1.06

glycosyltransferase

XAC0299

 

−2.16

polysaccharide /chitin deacetylase

XAC0575

ganB

−1.98

arabinogalactan endo-1,4-beta-galactosidase

XAC1286

abfA

−1.09

alpha-L-arabinofuranosidase

XAC1308

bga

−1.18

beta-galactosidase

XAC1309

galA

−1.49

arabinogalactan endo-1,4-beta-galactosidase

XAC1556

fucP

−1.43

glucose-galactose transporter

XAC1557

scrK

−1.49

fructokinase

XAC1558

 

−1.46

putative N-acylglucosamine 2-epimerase

XAC1770

celA

−1.03

cellulase

XAC1771

 

−1.02

sialic acid-specific 9-O-acetylesterase

XAC1793

celD

−2.46

glucan 1,4-beta-glucosidase

XAC1794

folk

−2.38

sodium/glucose cotransport protein

XAC1812

hmsF

−1.72

HmsF protein /Polysaccharide deacetylase

XAC1813

hmsH

−2.06

HmsH protein /substrate-specific transmembrane transporter

XAC2501

fruB

−1.73

multiphosphoryl transfer protein

XAC2502

fruK

−1.68

1-phosphofructokinase

XAC2503

fruA

−1.79

PTS system fructose-specific transporter subunit II

XAC3474

cit1

−1.08

citrate carrier protein

XAC3487

cebR

−2.20

transcriptional regulator

XAC3489

fyuA

−1.49

TonB-dependent sucrose outer membrane transporter

XAC3490

 

−1.22

amylosucrase or alpha amylase

XAC4195

ndvB/celAP

−1.23

NdvB protein/ cellobionic acid phosphorylase

XAC4355

 

−1.34

Glyco_hydro like

XAC4361

ttuB

−1.51

MFS transporter

Amino acid transport and metabolism

XAC0336

metE

1.72

5-methyltetrahydropteroyltriglutamate-methyltransferase

XAC0465

 

1.37

metalloproteinase

XAC1214

gcvP

1.09

glycine dehydrogenase

XAC2547

dapA

1.06

dihydrodipicolinate synthetase

XAC4326

uahA

6.50

urea amidolyase

XAC4327

uahA

5.92

allophanate hydrolase

XAC0174

phhA

−1.14

phenylalanine 4-monooxygenase

XAC0204

glnA

−3.39

glutamine synthetase

XAC0205

glnB

−3.01

nitrogen regulatory protein P-II

XAC0206

amtB

−2.78

ammonium transporter

XAC0300

pucG

−2.08

serine-pyruvate aminotransferase

XAC0301

amaB

−2.72

allantoate amidohydrolase

XAC1433

asnB

−1.19

asparagine synthetase B

XAC1820

thrA

−1.24

bifunctional aspartokinase I/homoserine dehydrogenase I

XAC1821

thrB

−1.20

homoserine kinase

XAC1823

thrC

−1.24

threonine synthase

XAC1828

hisG

−2.32

ATP phosphoribosyltransferase

XAC1829

hisD

−2.02

histidinol dehydrogenase

XAC1830

hisC

−1.94

histidinol-phosphate aminotransferase

XAC1831

hisB

−1.73

imidazole glycerol-phosphate dehydratase/phosphatase

XAC1832

hisH

−1.36

imidazole glycerol phosphate synthase subunit HisH

XAC1833

hisA

−1.61

1-(5-phosphoribosyl)-5- imidazole-4-carboxamide isomerase

XAC1834

hisF

−1.51

imidazole glycerol phosphate synthase subunit HisF

XAC1835

hisI

−1.12

phosphoribosyl-AMP cyclohydrolase

XAC3451

ilvC

−2.15

ketol-acid reductoisomerase

XAC3452

ilvG

−1.69

acetolactate synthase 2 catalytic subunit

XAC3453

ilvM

−1.49

acetolactate synthase isozyme II small subunit

XAC3454

tdcB

−1.71

threonine dehydratase

XAC3455

leuA

−1.22

2-isopropylmalate synthase

Lipid transport and metabolism

XAC0159

estA1

1.15

carboxylesterase type B

XAC1037

 

1.12

membrane protein

XAC1316

mmsB

1.03

3-hydroxyisobutyrate dehydrogenase

XAC0375

aes

−1.42

lipase

XAC2012

fadA

−1.25

acetyl-CoA acetyltransferase

XAC2013

fadB

−1.66

3-hydroxyacyl-CoA dehydrogenase

XAC3300

estA

−1.10

esterase

XAC3486

fabG

−3.14

3-ketoacyl-ACP reductase

XAC3959

 

−1.69

Acyl-CoA delta-9-desaturase

Inorganic ion transport and metabolism

XAC1578

phoX

1.34

phosphate-binding protein

XAC1579

oprO

1.50

polyphosphate-selective porin O

XAC0296

 

−2.50

monoxygenase

XAC0310

vanB

−3.94

vanillate O-demethylase oxidoreductase

XAC0311

vanA

−3.07

vanillate O-demethylase oxygenase

XAC0742

 

−1.45

RcnB containg protein

XAC0999

cirA

−1.04

colicin I receptor

XAC3168

bfeA

−1.55

ferric enterobactin receptor

XAC3169

bfeA

−1.17

ferric enterobactin receptor

XAC3472

oprO

−1.82

polyphosphate-selective porin O

XAC3484

oprO

−2.90

porin

For the genes involved in uptake and metabolism of amino acids, most (23 out of 29) were downregulated by DSF/Rpf QS in planta, while a small portion (6/29) were upregulated (Table 2). Among the downregulated genes, some are involved in the biosynthesis of asparagine (asnB/XAC1433), tyrosine (phhA/XAC0174), glutamine (glnA/XAC0204 and glnB/XAC0205), glycine (pucG/XAC0300 and amaB/XAC0301), threonine (thrAB/XAC1820, XAC1821, and thrC/XAC1823), histidine (hisGDCBHAFI/XAC1828–1835), and biosynthesis of isoleucine, leucine, and valine (ilvCGM, tdcB, leuA/XAC3451–3455). Over-presented in the up-regulated genes are those for biosynthesis of methionine (metE/XAC0306) and lysine (dapA/XAC2547), for a metalloproteinase (XAC0465), and for glycine biosynthesis and cleavage (gcvP/XAC1214). Remarkably, the urea amidolyase and an allophanate hydrolase, which catalyze the release of ammonia from urea, showed distinctive expression levels (Log2Fold Change ≥5.9) upregulated by DSF/RPF QS in Xac during host infection.

Eleven differentially expressed genes were related to inorganic ion transport and metabolism in Xac during host infection (Table 2). Remarkably, the two genes (phoX/ XAC1578 and oprO/XAC1579) encoding phosphate transporter proteins were upregulated by an average of 2.6-fold by DSF/Rpf QS during infection. The genes for siderophore biosynthesis (entF/XAC3922) and for iron storage protein in the bacterioferritin family (bfr/XAC1149) [42] were upregulated two-fold or more (Table 3). Six genes encoding TonB-dependent outer-membrane receptors involved in siderophore-mediated ferric iron uptake by Xac [42, 43], including fecA/XAC0690, btuB/XAC1310, and fyuA/XAC3489, were downregulated two-fold on average. In addition, the two genes coding for ferric enterobactin receptors involved in siderophore uptake (bfeA/ XAC3168 and XAC 3169) were also downregulated two-fold on average (Table 3).
Table 3

List of ferric iron uptake genes in Xac regulated by DSF/RPF during pathogenic process

Locus tag

Gene name

Log2Fold Change (Wt/∆rpfF)

Annotation/ Protein function

XAC1149

bfr

1.01

Bacterioferritin, iron storage

XAC3922

entF

1.42

ATP-dependent serine activating enzyme (nonribosomal peptide synthetases, siderophore biosyntensis)

XAC0690

fecA

−1.08

TonB-dependent outer membrane receptor

XAC1310

btuB

−2.07

TonB-dependent outer membrane receptor

XAC1768

fhuA

−1.19

TonB-dependent outer membrane receptor

XAC1769

cirA

−1.71

TonB-dependent outer membrane receptor

XAC2312

 

−1.27

TonB-dependent outer membrane receptor

XAC3489

fyuA

−1.49

TonB-dependent outer membrane receptor

XAC3168

bfeA

−1.55

Ferric enterobactin receptor, siderophore

XAC3169

bfeA

−1.18

Ferric enterobactin receptor, siderophore

Genes for signal transducers and/or transcriptional regulators regulated by DSF/Rpf-mediated QS in Xac during early stages of host infection

The expression of 12 genes coding for signal transducers and/or transcriptional regulators in Xac were differentially regulated by DSF/Rpf-mediated QS (Table 4). Of these, two genes were upregulated and 10 genes were downregulated. The two upregulated genes were XAC1328 and XAC3927, encoding a putative CheY-like superfamily protein and serine/threonine protein kinase respectively, both are of signal transducer activity. Among those genes downregulated were the two genes ntrB (XAC0207) and ntrC (XAC0208) encoding the NtrB/C two-component system, which interacts with the RpfC/G system responding to DSF signal to regulate sigma54-dependent promoters in vitro [44]. In addition, the two-component sensor genes tctE (XAC3482) and XAC3720, the transcriptional regulator genes acoR (XAC0654), tetR (XAC2014), iscR (XAC 2934), and cebR (XAC3487), the AbrB ambiactive repressor and activator (XAC1883), and the Trp operon repressor gene (trpR/XAC1827) were downregulated by the DSF/Rpf –mediated QS. The homologues of these signal transduction and transcription factors constitute regulators of virulence and adaptation factors in many bacteria, including the human bacterial pathogens enterotoxigenic E. coli [45] and P. aeruginosa [46], and the model organism Bacillus subtilis strain 168 [47]. For example, the IscR transcriptional repressor in E. coli negatively controls the type I fimbriae colonization factor synthesis and biofilm formation in response to both iron limitation and oxidative stress [45]. The trp repressor negatively regulates expression of genes involved in tryptophan biosynthesis, transport, and metabolism in response to intracellular levels of tryptophan, but also regulates transcription initiation in several other operons related to tryptophan metabolism that are important for expression of virulence factors in E. coli and P. aeruginosa [46]. Thus, they might function as regulators of virulence and adaptation factors in Xac by modulating biofilm formation and adhesion factor production, which are crucial for attachment and colonization of the tissues and for consequent invasion [17].
Table 4

Summary of Xac DEGs coding for signal transduction and transcriptional factors regulated by DSF/Rpf-mediated QS during pathogenic process

Locus tag

Gene name

Log2Fold Change (Wt/∆rpfF)

Annotation/ Protein function

XAC1328

 

1.07

CheY-like protein superfamily

XAC3927

 

1.04

serine/threonine protein kinase

XAC0207

ntrB

−1.28

two-component system sensor protein

XAC0208

ntrC

−1.21

two-component system regulatory protein

XAC0654

acoR

−1.27

transcriptional regulator AcoR

XAC1827

 

−2.41

hypothetical protein/ Trp repressor protein (represses transcription of the Trp operon)

XAC1883

 

−1.00

hypothetical protein/ AbrB domain containing transcriptional regulator

XAC2014

 

−1.29

TetR family transcriptional regulator

XAC2934

iscR

−1.02

hypothetical protein/ Iron-sulfur cluster regulator IscR (Fe-S assembly SUF system transcriptional regulator)

XAC3482

tctE

−1.02

two-component system sensor protein

XAC3487

cebR

−2.20

transcriptional regulator

XAC3720

 

−1.18

hypothetical protein/ putative two-component system sensor kinase

Putative function of the hypothetical protein encoding genes within the DSF/Rpf-mediated QS regulon in planta

BLASTx analysis showed that 39 of the 44 genes within the DSF/Rpf QS regulon encoding hypothetical proteins had significant similarities only to sequences in bacteria within the Xanthomonas genus. Based on sequence similarity and conserved domain detected, we defined putative functions for 24 of the 44 genes, which are potentially involved in bacterial adaptation and pathogenesis (Table 5). Some of these genes encode proteins with recognized roles in bacterial pathogenesis, such as members of the cell surface adhesion protein families (XAC3546) and chemotaxis protein families (XAC3753 and XAC3754). Interestingly, the genes encoding stress-induced protein (XAC2156) and Ferritin-like di-iron-carboxylate protein (XAC2155) were upregulated by DSF/Rpf –mediated QS and possibly involved in the adaptation of Xac to the host environment. The genes encoding putative GH18_chitinase-like glycosyl hydrolase (XAC3073) and GT2 family glycosyltransferase (XAC3533) were also upregulated, involved in carbohydrate transport and metabolism. In contrast, the gene XAC3085 encoding a putative T3SS effector protein was downregulated, with an unknown function in Xac-citrus interaction.
Table 5

Summary of Xac DEGs encoding hypothetical proteins regulated by DSF/Rpf-mediated QS during pathogenic process

Locus tag

Log2Fold Change (Wt/∆rpfF)

Homologue [Bacterial species]

Identity (%)c

XAC2155

1.36

ferritin-like domain-containing protein [Xanthomonas group]

99

XAC2156

1.97

stress-induced protein [X. phaseoli]

98

XAC3073

1.01

GH18_chitinase-like glycosyl hydrolase [X. citri]

99

XAC3533

1.23

glycosyltransferase, GT2 family [X. axonopodis]

97

XAC3546

1.29

autotransporter adhesion protein [X.citri]

99

XAC0295

−1.64

5-hydroxyisourate hydrolase [X. citri]

98

XAC0297

−2.93

2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase [X. citri]

99

XAC0298

−1.84

Nuclear transport factor 2 (NTF2-like) superfamily [X. axonopodis]

99

XAC0510

−1.22

FUSC-like inner membrane protein (fusaric acid resistance) [X. citri]

98

XAC1397

−2.05

Alginate export domain containing protein [X. axonopodis]

99

XAC1471

−1.12

Glycine zipper 2TM domain containing protein [X. citri]

98

XAC1827

−2.41

Trp repressor protein [Xanthomonas group]

99

XAC1883

−1.00

AbrB domain containing transcriptional regulator [X. citri]

99

XAC1884

−1.26

PIN (PilT N terminus) domain-containing protein [X. citri]

99

XAC2821

−1.02

Crotonase/Enoyl-Coenzyme A (CoA) hydratase [Xanthomonas group]

99

XAC2934

−1.02

Fe-S assembly SUF system transcriptional regulator [X. citri]

99

XAC3085

−1.06

putative type III secretion system effector protein [Xanthomonas group]

99

XAC3439

−1.16

putative secreted protein [Xanthomonas group]

99

XAC3506

−1.67

Cellulose belonging to glycosyl hydrolase family 5 [X. citri]

98

XAC3507

−1.99

CelS cellulose; Glycosyl hydrolase 12 superfamily [Xanthomonas group]

98

XAC3720

−1.17

putative two-component system sensor kinase [Xanthomonas group]

99

XAC3753

−1.22

putative chemotaxis membrane protein [Xanthomonas group]

99

XAC3754

−1.01

putative chemotaxis membrane protein [Xanthomonas group]

99

XAC3856

−1.19

calcium-binding protein, EFh Superfamily [X. citri]

99

XAC4219

−1.09

Lipid-binding SYLF domain containing protein [Xanthomonas group]

99

Comparison of the DSF/Rpf-mediated QS regulons in planta and in vitro

Our previous work identified 180 genes constituting the DSF/RpfF regulon of Xac grown in culture medium in the exponential and/or stationary growth phase [17]. Among those, a set of 31 genes were overlapping with the in planta DSF/Rpf regulon, 26 of which showed similar trends in alteration of expression between the two environmental conditions (Additional file 5: Table S5). Specifically, a subset of 20 genes were identified in the DSF/RpfF regulon of Xac in the exponential growth phase, 25 genes were identified in the DSF/RpfF regulon of Xac in the stationary growth phase, and 14 genes were identified in both regulons. These genes were primarily involved in energy metabolism (carbohydrate transport and metabolism), protein fate and protein synthesis (amino acid transport and metabolism or post-translational modification), and signal transduction or transcriptional regulation, and some encode hypothetical proteins with unknown functions.

Overview of citrus transcriptional responses to DSF/Rpf-mediated Xac infection

Global analyses of the citrus transcripts in response to DSF/Rpf-mediated Xac infection revealed that the protein families related to stress responses, signaling pathways, hormone metabolism, and cell wall modification were over-represented according to the gene ontology (GO) analysis (Fig. 4). Individual gene responses in metabolic pathways were visualized using the MapMan tool (Fig. 5). Remarkable downregulation was observed for many genes related to photosynthesis, secondary metabolism, and plant defense response.
Fig. 4

Gene Ontology classification of citrus differentially expressed genes (DEGs) response to the DSF/Rpf-mediated Xanthomonas citri subsp. citri infection. a Pie diagram depicting the relative abundance of each category of DEGs. The category was presented by functional classification followed by the corresponding percentage. b Column chart showing the relative abundance of the three main categories of DEGs: biological process, cellular component, and molecular function

Fig. 5

Display of citrus differentially expressed genes (DEGs) response to the DSF/Rpf-mediated Xanthomonas citri subsp. citri infection that are involved in different metabolic pathways (a) or biotic/abiotic stress responses (b). The log2 fold change of gene expression (∆rpfF -inoculated plants versus wild type Xanthomonas citri subsp. citri -inoculated plants) was analyzed using MapMan. Each square represents an individual gene within a category. Small squares colored in red and blue represent genes in infected plants that were up- and down-regulated by DSF/Rpf – mediated Xac infection, respectively. A false color scale was used and all the values were given on a log2 scale. The color saturates at a 4-fold change (i.e. log2 ratio = 2 or − 2). A significant downregulation was observed for many genes that are involved in photosynthesis, secondary metabolism, or response to biotic stress including genes for signaling, hormone metabolisms, and plant defense responses

DSF/Rpf -mediated Xac infection represses photosynthesis in citrus

The expression levels of nine genes involved in photosynthesis decreased significantly in wild type Xac strain 306 infected leaf tissues, compared with the ∆rpfF mutant infected leaf tissues (Table 6). Transcripts for photosystem II oxygen-evolving enhancer protein PsbO (Cs7g03508) and photosystem II 22 kDa protein PsbS (Cs3g19650) were less abundant in wild type Xac infected leaves. Three transcripts encoding subunits of photosystem I also decreased in wild type Xac infected leaves, including photosystem I reaction center subunit II (PsaD), VI-2 (PsaH), and O subunit (PsaO). In addition, the genes for photosynthetic electron transport protein plastocyanin (PetE) and for an ATP synthase subunit (the F-type H + −transporting ATPase subunit gamma, Cs4g10260) were downregulated in wild type Xac infected leaves (Table 6). These results are in agreement with the notion that Xac is biotrophic during early stages of host infection [48, 49] and that biotrophic pathogen infection generally represses photosynthesis in host plants [50].
Table 6

Summary of citrus DEGs genes involved in photosynthesis

ID

Gene name (Locus)

Log2Fold Change (Wt/∆rpfF)

Annotation/ Protein function

XLOC_017330

psbQ (Cs7g03580)

−1.36

photosystem II oxygen-evolving enhancer protein

XLOC_008489

psbS (Cs3g19650)

−2.10

photosystem II 22 kDa protein

XLOC_014472

psaD (Cs5g31180)

−1.18

photosystem I reaction center subunit II

XLOC_002098

psaH (Cs1g15170)

−1.14

photosystem I reaction center subunit VI-2

XLOC_015536

psaO (Cs6g12390)

−1.13

photosystem I subunit O

XLOC_008847

petE (Cs3g26730)

−1.22

photosynthetic electron transport protein plastocyanin

XLOC_004226

Cs2g26640

−1.27

GLK2 transcription factor, regulating the expression of photosynthetic apparatus

XLOC_010577

atpA (Cs4g10260)

−1.07

F-type H + −transporting ATPase subunit gamma

XLOC_001762

psaN (Cs1g09130)

1.23

photosystem I reaction center subunit N

Alterations of hormone metabolisms in citrus responding to DSF/Rpf-mediated Xac infection

Significant transcriptional changes in response to DSF/Rpf-mediated Xac infection were observed for a group of genes related to plant hormone biosynthesis, transportation, metabolism, and associated signal transduction (Table 7). Transcripts for auxin biosynthesis-related enzymes and auxin-responsive proteins, including indole-3-acetate beta-D-glucosyltransferase (IAGLU), UDP-glucosyltransferase (UGT74E2), and SAUR (small auxin-up RNA) -like auxin-responsive protein, were more abundant, while genes for the PIN or PIN-LIKES class of auxin transporters were downregulated in wild type Xac infected leaves. The gene Cs2g03270 encoding a 9-cis-epoxycarotenoid dioxygenase, a key enzyme for abscisic acid (ABA) biosynthesis [51], was downregulated in wild type Xac infected leaves, and gens for ABA-responsive (ABR) proteins were upregulated in wild type Xac infected leaves. Three genes involved in cytokinin biosynthesis (cytokinin synthase, isopentenyltransferase (IPT), and UDP-Glycosyltransferase superfamily protein) were downregulated in wild type Xac infected leaves, while two genes involved in cytokinin metabolic process (UDP-glucosyl transferase 85A5 (UGT85A5) and DON-Glucosyltransferase) were upregulated in wild type Xac infected leaves.
Table 7

Summary of citrus DEGs genes involved in plant hormone metabolisms

ID

Locus

Log2Fold Change (Wt/∆rpfF)

Annotation/ Protein function

Auxin biosynthesis, metabolism, and signaling

XLOC_012174

Cs5g20410

1.22

Indole-3-acetate beta-D-glucosyltransferase (IAGLU)

XLOC_031022

1.38

Indole-3-acetate beta-D-glucosyltransferase (IAGLU)

XLOC_005577

Cs2g23750

2.45

UDP-glucosyltransferase acting on IBA (indole-3-butyric acid), affects auxin homeostasis

XLOC_029081

orange1.1 t02620

1.65

SAUR-like auxin-responsive protein family

XLOC_003150

Cs2g06290

1.00

Aluminium induced protein with YGL and LRDR motifs, auxin-responsive

XLOC_015754

Cs6g17000

−1.61

Probable auxin efflux carrier component 1c (PIN1c)

XLOC_020295

Cs7g31320

−1.19

Auxin transporter-like protein 1 (PIN-like protein 1)

XLOC_008042

Cs3g10670

−1.28

NAD(P)-linked oxidoreductase superfamily protein, auxin regulated

Abscisic acid (ABA) -related genes

XLOC_004564

Cs2g03270

−1.21

9-cis-epoxycarotenoid dioxygenase for ABA biosynthesis

XLOC_004925

Cs2g10990

−1.71

UDP glycosyltransferase (UGT) for ABA biosynthesis

XLOC_017286

Cs7g02850

2.07

GRAM domain-containing protein, ABA-responsive protein-related

XLOC_017832

Cs7g13470

2.64

GRAM domain-containing protein, ABA-responsive protein-related

XLOC_012807

Cs5g32930

1.29

membrane-bound protein (Arabidopsis thaliana TSPO-related), induced by ABA

Ethylene - related genes

XLOC_010327

Cs4g05190

1.48

flavanone 3 hydroxylase, 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein, involved in ethylene synthesis

XLOC_004668

Cs2g05280

1.08

ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family (ERF1)

XLOC_014405

Cs5g29870

1.86

ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family (ERF1)

XLOC_024633

1.36

ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family(ERF1)

XLOC_007284

Cs3g23270

1.79

DREB subfamily A-5 of ERF/AP2 transcription factor family (RAP2.1)

XLOC_005573

Cs2g23660

− 1.31

Ethylene-responsive transcription factor 4 (Ethylene-responsive element-binding factor 4 homolog) (EREBP-3)

XLOC_003119

Cs2g05620

−1.32

Ethylene-responsive transcription factor 4 (Ethylene-responsive element-binding factor 4 homolog) (EREBP-3)

XLOC_001696

Cs1g07950

−1.17

ERF (ethylene response factor) subfamily B-1 of ERF/AP2 transcription factor family (ERF-4)

XLOC_001450

Cs1g03280

−1.07

ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family (ERF13)

XLOC_014725

−2.28

ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family (ERF-6)

XLOC_024283

Cs9g13620

−2.24

ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family (ERF104)

XLOC_023353

Cs9g13610

−2.04

ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family (ERF104)

XLOC_003353

Cs2g09980

−1.39

Ethylene-responsive nuclear protein / ethylene-regulated nuclear protein (ERT2)

XLOC_028605

orange1.1 t01663

−1.38

Adenine nucleotide alpha hydrolases-like superfamily protein, involved in response to stress

XLOC_002875

Cs2g01100

−1.97

DUF247 domain containing plant protein, probably involved in ethylene signal transduction

XLOC_004471

Cs2g01150

−1.43

DUF247 domain containing plant protein, probably involved in ethylene signal transduction

XLOC_004467

Cs2g01090

−1.01

DUF247 domain containing plant protein, probably involved in ethylene signal transduction

XLOC_014014

Cs5g22160

−1.18

DUF247 domain containing plant protein, probably involved in ethylene signal transduction

Cytokinin - related genes

XLOC_023917

Cs9g06010

−1.51

cytokinin synthase for cytokinin biosynthesis

XLOC_003491

Cs2g12620

−1.14

putative adenylate isopentenyltransferase (IPT), involved in cytokinin biosynthesis

XLOC_008154

Cs3g12960

− 1.59

UDP-Glycosyltransferase superfamily protein, involved in cytokinin biosynthesis

XLOC_030591

orange1.1 t05518

1.01

UDP-glucosyl transferase 85A5 (UGT85A5), involved in cytokinin metabolic process

XLOC_030963

1.47

DON-Glucosyltransferase, UDP-Glucosyl transferase superfamily protein, involved in cytokinin metabolic process

Gibberellic acid (GA)- related genes

XLOC_019477

Cs7g14940

−1.17

gibberellin 2-oxidase (GA2OX), involved in gibberellin metabolic process

XLOC_028715

orange1.1 t01909

−1.81

CYP701A cytochrome p450 family protein, involved in gibberellin biosynthesis

XLOC_005279

Cs2g17800

−1.57

ARM (Armadillo-type fold) repeat superfamily protein, involved in GA signal transduction

XLOC_005280

Cs2g17820

−1.14

ARM (Armadillo-type fold) repeat superfamily protein, involved in GAsignal transduction

XLOC_008817

Cs3g26100

−1.11

GA-responsive GAST like protein

XLOC_006493

Cs3g07395

1.16

Gibberellin-regulated family protein

Salicylic acid (SA) - related genes

XLOC_001130

Cs1g23160

1.00

Methyl salicylate (MeSA) esterase-like protein, involved in MeSA hydrolysis to SA

XLOC_005805

Cs2g28310

−1.04

S-adenosyl-L-methionine-dependent methyltransferases superfamily protein, involved in SA metabolic process

XLOC_016863

Cs6g18050

−1.33

S-adenosyl-L-methionine-dependent methyltransferases superfamily protein, involved in SA metabolic process

Jasmonic acid (JA) - related genes

 

XLOC_029628

orange1.1 t03726

1.35

12-oxophytodienoic acid reductases, involved in JA biosynthesis

XLOC_029630

orange1.1 t03729

1.64

FMN-containing oxidoreductases, involved in JA biosynthesis

XLOC_020298

Cs7g31430

1.05

S-adenosyl-L-methionine: jasmonic acid carboxyl methyltransferase (JMT), involved in JA metabolic process to form methyljasmonate (MeJA)

XLOC_026677

orange1.1 t03773

−1.51

Chloroplast lipoxygenase required for wound-induced JA accumulation in Arabidopsis

XLOC_029950

orange1.1 t04376

−2.04

Chloroplast lipoxygenase required for wound-induced JA accumulation in Arabidopsis

XLOC_002571

Cs1g24440

−1.22

S-adenosyl-L-methionine: jasmonic acid carboxyl methyltransferase (JMT), involved in JA metabolic process to form methyljasmonate (MeJA)

Brassinosteroid (BR) - related genes

XLOC_010301

Cs4g04730

−1.10

cycloartenol synthase 1 (CAS1), involved in the biosynthesis of BRs

XLOC_012247

Cs5g21830

−1.12

C-8 sterol isomerase, involved in the biosynthesis of BRs

XLOC_002765

− 2.43

Leucine-rich receptor-like protein kinase family protein, involved in BR signaling pathways

XLOC_006131

−1.52

Leucine-rich receptor-like protein kinase family protein, involved in BR signaling pathways

A total of 17 genes encoding the ethylene response factor (ERF) transcription factors were differentially expressed in wild type Xac infected leaves compared to ∆rpfF mutant infected leaves (Table 7). In particular, the transcripts for ERF1 and RAP2.1 were more abundant in wild type Xac infected leaves, while transcripts for EREBP-3, ERF-4, ERF-6, ERF104, and for an ethylene-regulated nuclear protein (ERT2) were less abundant in wild type Xac infected leaves. One gene (Cs4g05190) involved in ethylene biosynthesis was upregulated in wild type Xac infected leaves. Two genes for gibberellic acid (GA) biosynthesis (the CYP701A cytochrome p450 family protein) and GA inactivation (GA2OX: gibberellin 2-oxidase) [52] were downregulated in wild type Xac infected leaves (Table 7). Three genes involved in the GA response were also downregulated in wild type Xac infected leaves, including those GAST-like (gibberellic acid stimulated transcript-like) and ARM repeat superfamily proteins.

Three genes involved in jasmonic acid (JA) biosynthesis or metabolisms were upregulated in wild type Xac infected leaves compared to ∆rpfF mutant infected leaves (Table 7). These included the gene encoding 12-oxophytodienoic acid reductases (OPR) (orange1.1 t03726) and the gene encoding a FMN-containing oxidoreductases (orange1.1 t03729) (for JA biosynthesis), and a S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT) that catalyzes the formation of methyl jasmonate (MeJA) from JA (Cs7g31430) [53]. In contrast, two genes (orange1.1 t03773 and orange1.1 t04376) encoding the chloroplast lipoxygenases required for wound-induced JA accumulation in Arabidopsis were downregulated in wild type Xac infected leaves. Three genes involved in SA metabolisms were differentially expressed in wild type Xac infected leaves compared to ∆rpfF mutant infected leaves (Table 7). Notably, a gene (Cs1g23160) encoding the methyl esterase 1 (MES1) with methyl salicylate (MeSA) esterase activity of hydrolyzing MeSA to SA in planta [54], was upregulated in wild type Xac infected leaves. In addition, two genes (Cs2g28310 and Cs6g18050) encoding S-adenosyl-L-methionine-dependent methyl transferases superfamily proteins involved in SA metabolic process were downregulated in wild type Xac infected leaves. Furthermore, four genes involved in brassinosteroid (BR) biosynthesis or responses were repressed in wild type Xac infected leaves. They are a cycloartenol synthase 1 (CAS1) and a C-8 sterol isomerase involved in the biosynthesis of BR, and two leucine-rich receptor-like protein kinase family proteins involved in BR signaling pathways [55] (Table 7).

Citrus defense responses to DSF/Rpf-mediated Xac infection

Of the 1946 citrus DEGs between wild type Xac infected - and ∆rpfF mutant infected - libraries, 102 genes (5.4%) were identified to be involved in plant defense responses, with 32 genes upregulated and 70 genes downregulated by DSF/Rpf-mediated Xac infection (Additional file 6: Table S6; Table 8). Remarkably, 34 genes encoding plant immune receptor-like proteins or receptor-like kinases were downregulated. Eight genes encoding transcription regulators were downregulated, including three WRKY transcription factors- encoding genes (one for WRKY 4 and two for WRKY 53). In addition, four genes encoding pathogenesis-related (PR) family proteins were downregulated, including the genes encoding members of the PR-5 (thaumatin) and PR-6 (protease inhibitor) subfamily (Additional file 6: Table S6). Three Kunitz protease inhibitors encoding genes were also downregulated, which were suggested to modulate programmed cell death in in Arabidopsis during plant–pathogen interactions [56]. Other downregulated genes included five genes encoding NB-ARC (nucleotide-binding adaptor shared by Apaf-1, resistance proteins, and CED-4) domain-containing disease resistance proteins [57], and three genes encoding MYB transcription factor family proteins, which are involved in various plant biological processes including defense responses [58].
Table 8

Summary of citrus DEGs genes encoding putative immune receptors and transcription factors involved in plant defense responses

ID

Locus

Log2Fold Chang) (Wt/∆rpfF)

Annotation/ Protein function

Receptor encoding genes

 XLOC_030903

− 1.12

Receptor like protein 1 (RLP1), Leucine-rich repeat-containing

 XLOC_028463

orange1.1 t01371

−1.36

Receptor like protein 1 (RLP1), Leucine-rich repeat-containing

 XLOC_022248

Cs8g14810

−1.55

Receptor like protein 1 (RLP1), Leucine-rich repeat-containing

 XLOC_007802

Cs3g06050

−1.99

Receptor like protein 1 (RLP1), Leucine-rich repeat-containing

 XLOC_023272

Cs9g12160

−1.59

Receptor like protein 13 (RLP13), Leucine-rich repeat-containing

 XLOC_023274

Cs9g12220

−2.09

Receptor like protein 13 (RLP13), Leucine-rich repeat-containing

 XLOC_023264

Cs9g12040

−2.27

Receptor like protein 13 (RLP13), Leucine-rich repeat-containing

 XLOC_006617

Cs3g10050

−2.30

Receptor like protein 13 (RLP13), Leucine-rich repeat-containing

 XLOC_003292

−2.30

Receptor like protein 14 (RLP14), Leucine-rich repeat-containing

 XLOC_026146

orange1.1 t02820

−1.15

Receptor like protein 15 (RLP15), Leucine-rich repeat-containing

 XLOC_028464

orange1.1 t01372

−1.33

Receptor like protein 15 (RLP15), Leucine-rich repeat-containing

 XLOC_025415

orange1.1 t01415

−1.66

Receptor like protein 15 (RLP15), Leucine-rich repeat-containing

 XLOC_006615

Cs3g10010

−2.08

Receptor like protein 15 (RLP15), Leucine-rich repeat-containing

 XLOC_015519

Cs6g12110

−2.13

Receptor like protein 15 (RLP15), Leucine-rich repeat-containing

 XLOC_023261

Cs9g11990

−2.55

Receptor like protein 15 (RLP15), Leucine-rich repeat-containing

 XLOC_030349

orange1.1 t05075

−1.31

Receptor like protein 22 (RLP22), Leucine-rich repeat-containing

 XLOC_013590

Cs5g13820

−1.00

Receptor like protein 33 (RLP33), Leucine-rich repeat-containing

 XLOC_005928

Cs2g30850

−1.63

Receptor like protein 35 (RLP35), Leucine-rich repeat-containing

 XLOC_001914

Cs1g11900

−2.04

Receptor like protein 54 (RLP54), Leucine-rich repeat-containing

 XLOC_030467

orange1.1 t05273

−1.52

Receptor like protein 56 (RLP56), Leucine-rich repeat-containing

 XLOC_030282

orange1.1 t04923

−2.60

Receptor like protein 56 (RLP56), Leucine-rich repeat-containing

 XLOC_030151

orange1.1 t06047

−1.12

Receptor like protein 6 (RLP6), Leucine-rich repeat-containing

 XLOC_006431

Cs3g06220

−1.20

Receptor like protein 6 (RLP6), Leucine-rich repeat-containing

 XLOC_005373

Cs2g19490

−1.17

Receptor like protein 7 (RLP7), Leucine-rich repeat-containing

 XLOC_031340

−2.93

Receptor like protein 9 (RLP9), Leucine-rich repeat-containing

 XLOC_006131

−1.52

Receptor-like protein kinase family protein, Leucine-rich repeat-containing

 XLOC_012876

Cs5g34310

−1.09

Receptor-like protein kinase family protein, Leucine-rich repeat-containing

 XLOC_015528

Cs6g12270

−2.24

Receptor-like protein kinase family protein, Leucine-rich repeat-containing

 XLOC_002765

−2.43

Receptor-like protein kinase family protein, Leucine-rich repeat-containing

 XLOC_026533

orange1.1 t03518

−1.22

Disease resistance protein (TIR-NBS-LRR class) with transmembrane receptor activity

 XLOC_029900

orange1.1 t04292

−1.30

Disease resistance protein (TIR-NBS-LRR class) with transmembrane receptor activity

 XLOC_014032

Cs5g22400

−1.40

Disease resistance protein (TIR-NBS-LRR class) with transmembrane receptor activity

 XLOC_006408

Cs3g05870

−1.14

Disease resistance protein (CC-NBS-LRR class) family

 XLOC_006396

Cs3g05690

1.99

Disease resistance protein (TIR-NBS-LRR class) with transmembrane receptor activity

 XLOC_029612

orange1.1 t03700

1.14

Disease resistance protein (TIR-NBS-LRR class) with transmembrane receptor activity

 XLOC_006400

Cs3g05760

1.09

Disease resistance protein (TIR-NBS-LRR class) with transmembrane receptor activity

 XLOC_014130

Cs5g24240

1.08

Disease resistance protein (TIR-NBS-LRR class) with transmembrane receptor activity

Transcription factor encoding genes

 XLOC_013026

Cs5g03010

1.08

WRKY transcription factor family protein (WRKY22)

 XLOC_017469

Cs7g06330

1.17

WRKY transcription factor family protein (WRKY18)

 XLOC_016450

Cs6g10120

1.19

WRKY transcription factor family protein (WRKY54)

 XLOC_019872

Cs7g23080

1.46

MYB transcription factor family protein

 XLOC_020617

Cs8g02740

1.34

MYB transcription factor family protein

 XLOC_016421

Cs6g09420

−2.27

WRKY transcription factor family protein (WRKY 4)

 XLOC_028008

orange1.1 t00472

−1.26

WRKY transcription factor family protein (WRKY53)

 XLOC_013000

Cs5g02450

−1.04

WRKY transcription factor family protein (WRKY53)

 XLOC_005212

Cs2g16510

−2.28

MYB transcription factor family protein

 XLOC_014277

Cs5g27440

−1.82

MYB transcription factor family protein

 XLOC_024133

Cs9g10480

−1.54

MYB transcription factor family protein

 XLOC_011371

Cs5g04290

−1.52

Homeobox transcription factor family protein

 XLOC_013039

Cs5g03250

−1.29

Homeobox transcription factor family protein

 XLOC_021224

Cs8g14700

−1.66

NAC domain transcription factor family protein

 XLOC_021532

Cs8g21030

−1.08

NAC domain transcription factor family protein

 XLOC_025849

orange1.1 t0226

−1.32

RNA-binding (RRM/RBD/RNP motifs) family protein

Among the 32 genes upregulated by DSF/Rpf-mediated Xac infection, three genes encode WRKY transcription factors, including WRKY18, WRKY22, and WRKY54 (Table 8). Interestingly, in Arabidopsis, AtWRKY18 alone with AtWRKY40 and AtWRKY60, act as negative regulators of defense signaling [59]. Other upregulated genes include two genes coding for the MYB transcription factors, three genes for the PR family proteins including one PR-5 and two PR-6, four genes for the NB-LRR family receptors, five genes for wound-responsive or –induced proteins, and a few others for disease resistance responsive proteins and stress responsive proteins (Additional file 6: Table S6).

Expression of citrus genes associated with plant secondary metabolism and cell wall modification were altered by DSF/Rpf-mediated Xac infection

A total of 14 citrus genes related to the biosynthesis of flavonols, anthocyaninins, glucosinolates and terpenoids, which are well characterized defensive compounds [60], were downregulated by DSF/Rpf-mediated Xac infection (Additional file 7: Table S7). Five genes involved in lignin biosynthesis were upregulated by DSF/Rpf-mediated Xac infection, suggesting that lignin might be deposited in infected tissues, possibly as part of citrus responses to limit the pathogen colonization. Indeed, Xac infection induced the expression of genes related to lignin biosynthesis [61]; and, histological analyses revealed an increased lignin deposition and the existence of cell wall reinforcement in Xac infected tissues [62]. Remarkably, 12 genes encoding cell-wall-modifying enzymes, including expansins, endoglucanases, glycosyl transferases, and xyloglucan endotransglycosylases/hydrolases, were upregulated by DSF/Rpf-mediated Xac infection (Additional file 8: Table S8). Nine genes encoding protein products involved in the synthesis of cell wall precursors were also upregulated. These results implied a more pronounced effect on cell wall modification upon infection by the wild type Xac compared to the ∆rpfF mutant to limit the pathogen colonization.

Discussion

The in planta DSF/Rpf- mediated QS regulon of Xac

The results indicate that the DSF deficiency altered in planta expression of 202 genes in Xac, with a remarkable downregulation of different sets of genes functionally involved in stress tolerance, nutrition uptake and metabolisms, signal transduction, transcriptional regulation, and virulence. These findings support the hypothesis that the DSF/Rpf- mediated QS in Xac modulates diverse pathogenesis traits to promote bacterial adaptation to the host environment for a successful infection (Fig. 6). For example, Xac cells have to counteract environmental stresses and plant generated- oxidative stress during infection on citrus host [48, 63]. Our results showed that DSF/Rpf-mediated QS contributes to stress tolerance of Xac by positively regulating the expression of catalase, drug resistance translocase, defense protein-degrading endoproteinase, and the MFS drug transporter (Table 1). These enzymes are collectively important for bacterial resistance against diverse stresses from the environment and/or host organisms and thus for a successful infection [38, 64, 65]. DSF/Rpf-mediated QS also positively regulates the biosynthesis of trehalose, which protects Xac cells from osmotic and oxidative stresses to enable bacterial colonization in host plants [40], i.e., in the apoplast, an osmotic stressful environment [66].
Fig. 6

Hypothetical model of the modulation of citrus - Xanthomonas citri subsp. citri interactions by the DSF/Rpf- mediated quorum sensing (QS) during early stages of infection. Representative proteins and metabolic processes with important roles in plant-pathogen interactions are shown. Plant and bacterial molecules are depicted in light blue and red, respectively. DSF/Rpf- mediated QS modulates expression of diverse bacterial traits including adhesion, nutrition acquisition, stress tolerance (catalase and trehalose), signal transduction, transcription, and virulence factors, which collectively promote bacterial adaptation to the host environment to favor infection. The transcriptional alterations of citrus in response to DSF-mediated X. citri subsp. citri infection are characterized by the downregulation of photosynthesis, plant immune receptor-like proteins or receptor-like kinases including NBS-LRRs, NB-ARC resistance proteins, MYB and WRKY transcription factors, and pathogenesis-related (PR) proteins. Changes of phytohormone metabolism and signaling were also triggered by DSF-mediated X. citri subsp. citri infection, probably leading to increased accumulation of auxin, ethylene and jasmonic acid (JA), and decreased accumulation of brassinosteroid (BR), cytokinin and salicylic acid (SA), which may benefit the pathogen. Solid arrows with plus symbols indicate positive regulation and dashed arrows with minus symbols indicate negative regulation. Solid lines indicate information flow

The plant apoplast is low in nitrogen and rich in plant-derived sugars such as fructose [67]. Xac has adapted to the apoplast with diverse nutrient acquisition strategies evolved, including diverse enzymes for plant cell wall degradation, amino acid metabolism, carbohydrate metabolism and transportation [63]. The findings in this study indicate that Xac exploits the DSF/Rpf -mediated QS to regulate nutrition utilization during host infection (Table 2; Table 3). Interesting, the DSF/Rpf -mediated QS positively regulates the expression of phosphate transporter encoding genes, the homologues of which in X. axonopodis pv. glycines, the causal agent of bacterial pustule of soybean, have been demonstrated to be strongly expressed at early stages of infection and required for bacterial growth in host plants to promote disease [68]. DSF/Rpf-mediated QS also regulates ferric iron uptake of Xac in planta (Table 3). It has been reported that Xanthoferrin, a α-hydroxycarboxylate-type siderophore produced by Xcc is required for its optimum virulence [69]; and, DSF positively regulates the functions involved in ferric iron uptake to promote in planta growth of X. oryzae pv. oryzicola [70]. However, there is no evidence that iron is limited or available to Xac cells grown in planta. The functional role of DSF/Rpf regulated ferric iron uptake in Xac biology and pathogenesis remains to be determined.

Importantly, the DSF/Rpf-mediated QS differentially regulated the expression of 12 determined or putative signal transducers and/or transcriptional regulators, most of which were downregulated, including the NtrB/C two-component system (Table 4). The NtrB/C system interacts with the RpfC/G system in responding to DSF signal to regulate sigma54-dependent promoters in Xac in vitro [44]. Our findings suggested that the DSF signal negatively regulates sigma54-dependent promoters through the RpfCG- NtrBC- sigma54 pathway in Xac during early stages of host infection. The functional roles of the other signal transducers and/or transcriptional regulators regulated by the DSF/Rpf-mediated QS remain unknown. Collectively, the results suggested that DSF-mediated signaling might be linked with diverse regulators to enable complex patterns of gene expression to be employed by Xac to favor infection in host plants, which deserves further investigations.

Comparison of the in planta and in vitro DSF/RpfF regulons revealed that a set of 31 genes were commonly differentially regulated by DSF/RpfF under the two environment conditions. There are a large number of unique genes in the in planta regulon that were not regulated by DSF/RpfF in vitro (Additional file 5: Table S5). A couple of reasons could explain the differences among the in planta and in vitro DSF/RpfF regulons. It could be because of the difference in cell density of Xac in the two experimental conditions: approximately 108 CFU/cm2 of leaf tissues for in planta experiments (Fig. 1b) and 109 to 1010 CFU/ml of growth medium for in vitro experiments [17], as the QS regulates expression of genes in a cell density- dependent manner. It also could be because that the DSF/Rpf – mediated QS might play divers roles in regulating gene expression of Xac under different environment conditions. Several subsets of unique genes within the in planta regulon that were downregulated are involved in cell surface adhesion, stress tolerance, carbohydrate transport and metabolism, amino acids uptake and metabolism, signal transduction, and transcriptional regulation, which are in agreement with the findings produced in analysis of DSF/Rpf in vitro regulon [17]. The regulation pattern of Xac in planta compared to in vitro indicates the needs for real-time and in situ studies.

Citrus transcriptional responses to DSF/Rpf-mediated Xac infection

Gene expression data indicated that significant transcriptional alterations occurred in citrus plants in response to DSF/Rpf-mediated Xac infection, which caused various changes in plant immunity and physiology, thus favoring the pathogen infection. Especially, a large group of genes differentially expressed, related to plant hormone biosynthesis, transportation, metabolism, and associated signal transduction (Table 7). The results suggested the existence of elevated levels of auxin in wild type Xac infected leaves compared with the ∆rpfF mutant infected leaves. Auxin has been shown to promote citrus canker development [71]; and auxin pathways play a role in tomato bacterial wilt caused by Ralstonia solanacearum [72]. Therefore, it is likely that the alterations in expression of auxin biosynthesis, mobilization and signaling genes in response to the DSF/Rpf-mediated Xac infection are associated with the citrus canker disease development. Additionally, cytokinin biosynthesis genes were downregulated and cytokinin metabolic genes were upregulated, implying decreased accumulation of cytokinin in wild type Xac infected leaves. Cytokinin has been shown to regulate plant defense responses in a dosage-dependent manner: strong activation of cytokinin signaling confers resistance to biotrophic pathogens via increased SA accumulation; by contrast, weak activation of cytokinin signaling suppresses pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) [73]. Our results suggested that the DSF/Rpf-mediated Xac infection modulates cytokinin accumulation and thus avoids strong activation of cytokinin signaling to promote host susceptibility. Another interesting finding is the upregulation of genes involved in the biosynthesis of and response to ethylene in wild type Xac infected leaves. Xac infection activates ethylene biosynthesis and signaling in citrus plants [61]. Ethylene is usually involved in plant defense responses against necrotrophic pathogens [74], thus it is possible that the successful establishment of Xac infection is favored by the development of inadequate plant defenses.

Notably, genes for JA biosynthesis and for SA production (i.e., the MeSA esterase) were upregulated in wild type Xac infected leaves, while genes for SA metabolic process and for BR biosynthesis or responses were downregulated (Table 7). Earlier reports showed that certain antagonistic relationships occur between BR and JA, JA and SA pathways, and BR signaling negatively regulates plant defense against pathogens [55, 75, 76]. Both biotrophic and hemibitrophic pathogens employ the antagonism between JA and SA pathways and activate JA signaling to promote infection [77, 78]. The findings in this study implied that the DSF/Rpf-mediated Xac infection may activate the JA signaling pathway and repress BR signaling to benefit the pathogen during early stages of infection.

Gene expression levels point to that the activity of DSF/Rpf-mediated QS might induce plant basal defenses and repress secondary defenses of citrus to promote Xac infection (Table 8; Additional file 6: Table S6; Additional file 7: Table S7; Additional file 8: Table S8). Remarkably, many plant immune receptor -like proteins or receptor-like kinases proteins were downregulated by DSF/Rpf-mediated Xac infection (Table 8), which are believed to perceive extracellular molecules, including microbe/pathogen-associated molecular patterns (M/PAMP) and environmental stimuli to induce plant basal resistance [79, 80]. In addition, four putative NB-LRR family proteins were also downregulated by DSF/Rpf-mediated Xac infection, which are intracellular proteins and recognize pathogen effectors to lead to strong resistance responses [81]. Overall, it is important to note that more defense- related genes were downregulated than upregulated by DSF/Rpf-mediated Xac infection (70 downregulated versus 32 upregulated) (Additional file 6: Table S6), especially in the group of immune receptors (34 downregulated versus 4 upregulated) (Table 8).

It is not clear how the activity of DSF/Rpf-mediated QS triggers plant basal defenses and represses secondary defenses of citrus plants. One possible explanation might lie in the observations that the DSF signal molecule itself could elicit plant defense response in Xanthomonas–host plant interactions and wild-type Xanthomonas spp. can suppress the DSF-induced defense responses by the production of the EPS xanthan and T3SS effectors [18]. Our results showed that the DSF/Rpf-mediated QS did not regulate or affect the production of the EPS xanthan by Xac in citrus during early stages of infection, but negatively regulated the expression of a putative T3SS effector (XAC3085) (Table 5). The homologue of XAC3085 in X. campestris pv. vesicatoria (also termed X. euvesicatoria), the causal agent of bacterial spot disease on pepper and tomato, was determined as a T3SS effector named XopK, whose function remains unknown but seems not to contribute to the virulence of the pathogen [82]. Xac might suppress the DSF molecule elicited plant defense responses through the EPS and/or the T3SS effectors that are not affected by the DSF/Rpf-mediated QS during host infection. Another possible reason might be the functional interplay between the bacterial T2SS and T3SS in modulating plant defense responses and promoting disease as observed in the X. oryzae pv. oryzae – rice interactions, where the bacterial T2SS secreted virulence factors: the ClsA cellulase and CbsA cellobiosidase, induced innate rice defense responses that were suppressed by T3S effectors [83]. We found that the DSF/Rpf-mediated QS positively regulates the expression of the homologue (engXCA/XAC0612) of the ClsA cellulase (Table 2). Therefore, wild-type Xac may suppress, in a T3SS-dependent manner, the citrus plant defense responses probably induced by the T2SS effector cellulase (engXCA/XAC0612) to enable successful infection.

Conclusions

In conclusion, this work provides an in-depth transcriptomic analysis of DSF/Rpf -mediated QS regulation from both pathogen and host sides during the biotrophic interactions between Xac and citrus. Based on the results obtained, a model was presented that describes the major molecular and physiological aspects regulated by the DSF/Rpf- mediated QS during early stages of infection (Fig. 6). The findings support the hypothesis that the DSF/Rpf- mediated QS in Xac modulates diverse pathogenesis traits to promote bacterial adaptation to the host environment, and triggers various changes in plant immunity and physiology favoring the pathogen for successful infection. Taken together, the present work has provided novel insights into the role of the DSF/Rpf- mediated QS regulatory system in the pathogenic interactions between Xanthomonas and its host plants and expanded our current knowledge of DSF- mediated QS regulation, and adds to our general understanding of plant-pathogen interactions.

Notes

Acknowledgments

The authors would like to thank Zgiqian Pang and Doron Temper for insightful discussions.

Funding

This work was supported by Florida Citrus Research and Development Foundation and the US Department of Agriculture-National Institute of Food and Agriculture (USDA-NIFA) Plant Biotic Interactions Program 2017–67013-26527 (to NW), and the China Scholarship Council (CSC) awarded (to LL).

Availability of data and materials

The RNA sequence dataset supporting the results in this article is available from the NCBI under the bioproject no. PRJNA421992 with the SRA accession no. SRP126698 (https://www.ncbi.nlm.nih.gov//sra/?term=SRP126698).

Author’s contributions

NW, JL, and LL conceived and designed the experiments. LL and JL performed the experiments. LL, JL, YZ, and NW analyzed data. LL, JL, YZ, and NW wrote the manuscript. All authors read and approved the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary material

12864_2018_5384_MOESM1_ESM.docx (19 kb)
Additional file 1: Table S1. Primers used for qRT-PCR assays for experimental validation (DOCX 18 kb)
12864_2018_5384_MOESM2_ESM.docx (13 kb)
Additional file 2: Table S2. Summary of the RNA-seq data (DOCX 13 kb)
12864_2018_5384_MOESM3_ESM.docx (35 kb)
Additional file 3: Table S3. Detail of the DEGs of Xanthomonas citri subsp. citri regulated by DSF/RpfF –mediated QS (DOCX 35 kb)
12864_2018_5384_MOESM4_ESM.xlsx (183 kb)
Additional file 4: Table S4. Detail of the DEGs of citrus in response to DSF/RpfF –mediated Xac infection (XLSX 183 kb)
12864_2018_5384_MOESM5_ESM.xlsx (29 kb)
Additional file 5: Table S5. Comparison of the in vitro and in planta DSF/Rpf-mediated QS regulons of Xanthomonas citri subsp. citri (XLSX 29 kb)
12864_2018_5384_MOESM6_ESM.xlsx (18 kb)
Additional file 6: Table S6. Differentially expressed citrus genes related to plant defense responses (XLSX 17 kb)
12864_2018_5384_MOESM7_ESM.xlsx (11 kb)
Additional file 7: Table S7. Differentially expressed citrus genes involved in plant secondary metabolisms (XLSX 11 kb)
12864_2018_5384_MOESM8_ESM.xlsx (14 kb)
Additional file 8: Table S8. Differentially expressed citrus genes involved in cell wall modifications (XLSX 13 kb)

References

  1. 1.
    Büttner D, Bonas U. Regulation and secretion of Xanthomonas virulence factors. FEMS Microbiol Rev. 2010;34:107–33.CrossRefGoogle Scholar
  2. 2.
    Ryan RP, Vorhoelter FJ, Potnis N, Jones JB, Van Sluys MA, Bogdanove AJ, et al. Pathogenomics of Xanthomonas: understanding bacterium-plant interactions. Nat Rev Microbiol. 2011;9:344–55.CrossRefGoogle Scholar
  3. 3.
    Strauß T, van Poecke RMP, Strauß A, Röme P, Minsavage GV, et al. RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome. Proc Natl Acad Sci U S A. 2012;109(47):19480–5.CrossRefGoogle Scholar
  4. 4.
    Zhou X, Hu X, Li J, Wang N. A novel periplasmic protein, VrpA, contributes to efficient protein secretion by the type III secretion system in Xanthomonas spp. Mol Plant-Microbe Interact. 2015;28(2):143–53.CrossRefGoogle Scholar
  5. 5.
    Li J, Wang N. The gpsX gene encoding a glycosyltransferase is important for polysaccharide production and required for full virulence in Xanthomonas citri subsp. citri. BMC Microbiol. 2012;12:31.CrossRefGoogle Scholar
  6. 6.
    Ryan RP, An SQ, Allan JH, McCarthy Y, Dow JM. The DSF-family of cell-cell signals: An expanding class of bacterial virulence regulators. PLoS Pathog. 2015;11:e1004986.CrossRefGoogle Scholar
  7. 7.
    Dow JM. Diffusible signal factor-dependent quorum sensing in pathogenic bacteria and its exploitation for disease control. J Appl Microbiol. 2017;122(1):2–11.CrossRefGoogle Scholar
  8. 8.
    Wang LH, He YW, Gao YF, Wu JE, Dong YH, He C, et al. A bacterial cell-cell communication signal with cross-kingdom structural analogues. Mol Microbiol. 2004;51:903–12.CrossRefGoogle Scholar
  9. 9.
    Tang JL, Liu YN, Barber CE, Dow JM, Wootton JC, et al. Genetic and molecular analsysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris. Mol Gen Genet. 1991;226:409–17.CrossRefGoogle Scholar
  10. 10.
    Slater H, Alvarez-Morales A, Barber CE, Daniels MJ. Dow JM. A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris. Mol Microbiol. 2000;38:986–1003.CrossRefGoogle Scholar
  11. 11.
    Wang XY, Zhou L, Yang J, Ji GH, He YW. The RpfB-dependent quorum sensing signal turnover system is required for adaptation and virulence in rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae. Mol Plant-Microbe Interact. 2016;29(3):220–30.CrossRefGoogle Scholar
  12. 12.
    Ryan RP, Fouhy Y, Lucey JF, Crossman LC, Spiro S, He YW, et al. Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc Natl Acad Sci U S A. 2006;103:6712–7.CrossRefGoogle Scholar
  13. 13.
    Ryan RP, McCarthy Y, Andrade M, Farah CS, Armitage JP, Dow JM. Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris. Proc Natl Acad Sci U S A. 2010;107:5989–94.CrossRefGoogle Scholar
  14. 14.
    Cai Z, Yuan ZH, Zhang H, Pan Y, Wu Y, Tian XQ, et al. Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence. PLoS Pathog. 2017;13(4):e1006304.CrossRefGoogle Scholar
  15. 15.
    Dow JM, Crossman L, Findlay K, He YQ, Feng JX, Tang JL. Biofilm dispersal in Xanthomonas campestris is controlled by cell-cell signaling and is required for full virulence to plants. Proc Natl Acad Sci U S A. 2003;100:10995–00.CrossRefGoogle Scholar
  16. 16.
    An SQ, Allan JH, McCarthy Y, Febrer M, Dow JM, et al. The PAS domain-containing histidine kinase RpfS is a second sensor for the diffusible signal factor of Xanthomonas campestris. Mol Microbiol. 2014;92:586–97.CrossRefGoogle Scholar
  17. 17.
    Guo Y, Zhang Y, Li JL, Wang N. Diffusible signal factor-mediated quorum sensing plays a central role in coordinating gene expression of Xanthomonas citri subsp citri. Mol Plant-Microbe Interact. 2012;25:165–79.CrossRefGoogle Scholar
  18. 18.
    Kakkar A, Nizampatnam NR, Kondreddy A, Pradhan BB, Chatterjee S. Xanthomonas campestris cell-cell signaling molecule DSF (diffusible signal factor) elicits innate immunity in plants and is suppressed by the exopolysaccharide xanthan. J Exp Bot. 2015;66:6697–714.CrossRefGoogle Scholar
  19. 19.
    Deng Y, Wu J, Yin W, Li P, Zhou J, Chen S, et al. Diffusible signal factor family signals provide a fitness advantage to Xanthomonas campestris pv. campestris in interspecies competition. Environ Microbiol. 2016;18:1534–45.CrossRefGoogle Scholar
  20. 20.
    He YW, Ng AY, Xu M, Lin K, Wang LH, Dong YH, Zhang LH. Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network. Mol Microbiol. 2007;64:281–92.CrossRefGoogle Scholar
  21. 21.
    An SQ, Febrer M, McCarthy Y, Tang DJ, Clissold L, Kaithakottil G, et al. High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis. Mol Microbiol. 2013;88:1058–69.CrossRefGoogle Scholar
  22. 22.
    O'Connell A, An SQ, McCarthy Y, Schulte F, Niehaus K, et al. Proteomics analysis of the regulatory role of Rpf/DSF cell-to-cell signaling system in the virulence of Xanthomonas campestris. Mol Plant-Microbe Interact. 2013;26:1131–7.CrossRefGoogle Scholar
  23. 23.
    Zhao Y, Qian G, Yin F, Fan J, Zhai Z, Liu C, Hu B, Liu F. Proteomic analysis of the regulatory function of DSF-dependent quorum sensing in Xanthomonas oryzae pv. oryzicola. Microb Pathogenesuis. 2011;50:48–55.CrossRefGoogle Scholar
  24. 24.
    da Silva AC, Ferro JA, Reinach FC, Farah CS, Furlan LR, Quaggio RB, et al. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature. 2002;417:459–63.CrossRefGoogle Scholar
  25. 25.
    Langmead B, Salzberg SL. Fast gapped-read alignment with bowtie 2. Nat Methods. 2012;9:357–U54.CrossRefGoogle Scholar
  26. 26.
    Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.CrossRefGoogle Scholar
  27. 27.
    Trapnell C, Roberts A, Goff L, Pertea G, Kim D, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and cufflinks. Nat Protoc. 2012;7(3):562–78.CrossRefGoogle Scholar
  28. 28.
    Xu Q, Chen LL, Ruan X, Chen D, Zhu A, Chen C, et al. The draft genome of sweet orange (Citrus sinensis). Nat Genet. 2013;45:59–66.CrossRefGoogle Scholar
  29. 29.
    Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36.CrossRefGoogle Scholar
  30. 30.
    Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol. 2013;31(1):46–53.CrossRefGoogle Scholar
  31. 31.
    Lohse M, Nagel A, Herter T, May P, Schroda M, Zrenner R, et al. Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data. Plant Cell Environ. 2014;37(5):1250–8.CrossRefGoogle Scholar
  32. 32.
    Thimm O, Bläsing O, Gibon Y, Nagel A, Meyer S, Krüger P, et al. MapMan: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J. 2004;37(6):914–39.CrossRefGoogle Scholar
  33. 33.
    Dash S, Hemert JV, Hong L, Wise RP, Dickerson JA. PLEXdb: gene expression resources for plants and plant pathogens. Nucleic Acids Res. 2012;40(D1):D1194–201.CrossRefGoogle Scholar
  34. 34.
    Du Z, Zhou X, Ling Y, Zhang Z, Su Z. agriGO: A GO analysis toolkit for the agricultural community. Nucleic Acids Res 2010; gkq310.  https://doi.org/10.1093/nar/gkq310.
  35. 35.
    Fan J, Chen C, Yu Q, Khalaf AA, Achor DS, Brlansky RH, et al. Comparative transcriptional and anatomical analyses of tolerant rough lemon and susceptible sweet orange in response to ‘Candidatus Liberibacter asiaticus’ infection. Mol Plant-Microbe Interact. 2012;25:1396–07.CrossRefGoogle Scholar
  36. 36.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods. 2001;25(4):402–8.CrossRefGoogle Scholar
  37. 37.
    Engel LS, Hill JM, Caballero AR, Green LC, O'Callaghan RJ. Protease IV, a unique extracellular protease and virulence factor from Pseudomonas aeruginosa. J Biol Chem. 1998;273:16792–7.CrossRefGoogle Scholar
  38. 38.
    Park S-J, Kim S-K, So Y-I, Park H-Y, Li X-H, et al. Protease IV, a quorum sensing-dependent protease of Pseudomonas aeruginosa modulates insect innate immunity. Mol Microbiol. 2014;94:1298–14.CrossRefGoogle Scholar
  39. 39.
    Kurz M, Burch AY, Seip B, Lindow SE, Gross H. Genome-driven investigation of compatible solute biosynthesis pathways of Pseudomonas syringae pv. syringae and their contribution to water stress tolerance. Appl Environ Microbiol. 2010;76(16):5452–62.CrossRefGoogle Scholar
  40. 40.
    Piazza A, Zimaro T, Garavaglia BS, Ficarra FA, Thomas L, Marondedze C, et al. The dual nature of trehalose in citrus canker disease: a virulence factor for Xanthomonas citri subsp. citri and a trigger for plant defence responses. J Exp Bot. 2015;66(9):2795–11.CrossRefGoogle Scholar
  41. 41.
    Tondo ML, Petrocelli S, Ottado J, Orellano EG. The monofunctional catalase KatE of Xanthomonas axonopodis pv. citri is required for full virulence in citrus plants. PLoS One. 2010;5(5):e10803.CrossRefGoogle Scholar
  42. 42.
    Etchegaray A, Silva-Stenicoa ME, Moona DH, Tsaia SM. In silico analysis of nonribosomal peptide synthetases of Xanthomonas axonopodis pv. citri: identification of putative siderophore and lipopeptide biosynthetic genes. Microbiol Res. 2004;159:425–37.CrossRefGoogle Scholar
  43. 43.
    Wiggerich H-G, Puhler A. The exbD2 gene as well as the iron-uptake genes tonB, exbB and exbD1 of Xanthomonas campestris pv. campestris are essential for the induction of a hypersensitive response on pepper (Capsicum annuum). Microbiology. 2000;146:1053–60.CrossRefGoogle Scholar
  44. 44.
    Andrade MO, Alegria MC, Guzzo CR, Docena C, Rosa MCP, Ramos CHI, Farah CS. The HD-GYP domain of RpfG mediates a direct linkage between the Rpf quorum-sensing pathway and a subset of diguanylate cyclase proteins in the phytopathogen Xanthomonas axonopodis pv citri. Mol Microbiol. 2006;62(2):537–51.CrossRefGoogle Scholar
  45. 45.
    Haines S, Arnaud-Barbe N, Poncet D, Reverchon S, Wawrzyniak J, Nasser W, Renauld-Mongénie G. IscR regulates synthesis of colonization factor antigen I fimbriae in response to iron starvation in enterotoxigenic Escherichia coli. J Bacteriol. 2015;197:2896–07.CrossRefGoogle Scholar
  46. 46.
    Merino E, Jensen RA, Yanofsky C. Evolution of bacterial trp operons and their regulation. Curr Opin Microbiol. 2008;11(2):78–86.CrossRefGoogle Scholar
  47. 47.
    Schultz D, Wolynes PG, Jacob EB, Onuchic JN. Deciding fate in adverse times: sporulation and competence in Bacillus subtilis. Proc Natl Acad Sci U S A. 2009;106:21027–34.CrossRefGoogle Scholar
  48. 48.
    Graham JH, Gottwald TR, Cubero J, Achor DS. Xanthomonas axonopodis pv. citri: factors affecting successful eradication of citrus canker. Mol Plant Pathol. 2004;5:1–15.CrossRefGoogle Scholar
  49. 49.
    Gottig N, Garavaglia BS, Daurelio LD, Valentine A, Gehring C, Orellano EG, Ottado J. Xanthomonas axonopodis pv. citri uses a plant natriuretic peptide-like protein to modify host homeostasis. Proc Natl Acad Sci U S A. 2008;105:18631–6.CrossRefGoogle Scholar
  50. 50.
    Berger S, Benediktyova Z, Matous K, Bonfig K, Mueller MJ, Roitsch T. Visualization of dynamics of plant-pathogen interaction by novel combination of chlorophyll fluorescence imaging and statistical analysis: differential effects of virulent and avirulent strains of P. syringae and of oxylipins on A. thaliana. J Exp Bot. 2007;58:797–06.CrossRefGoogle Scholar
  51. 51.
    Iuchi S, Kobayashi M, Taji T, Naramoto M, Seki M, Kato T, et al. Regulation of drought tolerance by gene manip-ulation of 9-cis-epoxycarotenoid dioxygenase: a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 2001;27:325–33.CrossRefGoogle Scholar
  52. 52.
    Thomas SG, Phillips AL, Hedden P. Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation. Proc Natl Acad Sci U S A. 1999;96:4698–03.CrossRefGoogle Scholar
  53. 53.
    Turner JG, Ellis C, Devoto A. The Jasmonate signal pathway. Plant Cell. 2002;14(Suppl):s153–64.CrossRefGoogle Scholar
  54. 54.
    Park SW, Kaimoyo E, Kumar D, Mosher S, Klessig DF. Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science. 2007;318:113–6.CrossRefGoogle Scholar
  55. 55.
    Lozano-Duran R, Zipfel C. Trade-off between growth and immunity: role of brassinosteroids. Trends Plant Sci. 2015;20:12–9.CrossRefGoogle Scholar
  56. 56.
    Li J, Brader G, Palva ET. Kunitz trypsin inhibitor: An antagonist of cell death triggered by Phytopathogens and Fumonisin B1 in Arabidopsis. Mol Plant. 2008;1(3):482–95.CrossRefGoogle Scholar
  57. 57.
    Qi D, Innes RW. Recent advances in plant NLR structure, function, localization and signaling. Front Immunol. 2013.  https://doi.org/10.3389/fimmu.2013.00348.
  58. 58.
    Ambawat S, Sharma P, Yadav NR, Yadav RC. MYB transcription factor genes as regulators for plant responses: an overview. Physiol Mol Biol Plants. 2013;19(3):307–21.CrossRefGoogle Scholar
  59. 59.
    Pandey SP, Somssich IE. The role of WRKY transcription factors in plant immunity. Plant Physiol. 2009;150(4):1648–55.CrossRefGoogle Scholar
  60. 60.
    Michael P, Speed M, Fenton A, Jones MG, Ruxton GD, Brockhurst MG. Coevolution can explain defensive secondary metabolite diversity in plants. New Phytol. 2015;208(4):1251–66.CrossRefGoogle Scholar
  61. 61.
    Cernadas RA, Camillo LR, Benedetti C. Transcriptional analysis of the sweet orange interaction with the citrus canker pathogens Xanthomonas axonopodis pv. citri and Xanthomonas axonopodis pv. aurantifolii. Mol Plant Pathol. 2008;9(5):609–31.CrossRefGoogle Scholar
  62. 62.
    Kraiselburd I, Daurelio LD, Tondo ML, Merelo P, Cortadi AA, Talón M, et al. The LOV protein of Xanthomonas citri subsp. citri plays a significant role in the counteraction of plant immune responses during citrus canker. PLoSONE. 2013;8(11):e80930.CrossRefGoogle Scholar
  63. 63.
    Yan Q, Wang N. High-throughput screening and analysis of genes of Xanthomonas citri subsp. citri involved in citrus canker symptom development. Mol Plant-Microbe Interact. 2012;25(1):69–84.CrossRefGoogle Scholar
  64. 64.
    Du D, Van VHW MS, Pos KM, Luisi BF. Structure, mechanism and cooperation of bacterial multi drug transporters. Curr. Opin. Struct Biol. 2015;33:76–91.Google Scholar
  65. 65.
    Quistgaard EM, Löw C, Guettou F, Nordlund P. Understanding transport by the major facilitator superfamily (MFS): structures pave the way. Nat Rev Mol Cell Biol. 2016;17:123–32.CrossRefGoogle Scholar
  66. 66.
    Yu X, Lund SP, Scott RA, Greenwald JW, Records AH, Nettleton D, et al. Transcriptional responses of Pseudomonas syringae to growth in epiphytic versus apoplastic leaf sites. Proc Natl Acad Sci U S A. 2013;110:E425–34.CrossRefGoogle Scholar
  67. 67.
    Rico A, Preston GM. Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast-induced nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast. Mol Plant-Microbe Interact. 2008;21(2):269–82.CrossRefGoogle Scholar
  68. 68.
    Chatnaparat T, Prathuangwong S, Lindow SE. Global pattern of gene expression of Xanthomonas axonopodis pv. glycines within soybean leaves. Mole Plant-Microbe Interact. 2016;29(6):508–22.CrossRefGoogle Scholar
  69. 69.
    Pandey SS, Patnana PK, Rai R, Chatterjee S. Xanthoferrin, the α-hydroxycarboxylate-type siderophore of Xanthomonas campestris pv. campestris, is required for optimum virulence and growth inside cabbage. Mol Plant Pathol. 2017;18(7):949–62.CrossRefGoogle Scholar
  70. 70.
    Rai R, Javvadi S, Chatterjee S. Cell–cell signalling promotes ferric iron uptake in Xanthomonas oryzae pv. oryzicola that contribute to its virulence and growth inside rice. Mol Microbiol. 2015;96:708–27.CrossRefGoogle Scholar
  71. 71.
    Cernadas A, Benedetti CE. Role of auxin and gibberellin in citrus canker development and in the transcriptional control of cell-wall remodeling genes modulated by Xanthomonas axonopodis pv citri. Plant Sci. 2009;177(3):190–5.CrossRefGoogle Scholar
  72. 72.
    French E, Kim B-S, Rivera-Zuluaga K, Iyer-Pascuzzi A. Whole root transcriptomic analysis suggests a role for auxin pathways in resistance to Ralstonia solanacearum in tomato. Mol Plant-Microbe Interact. 2018;31(4):432–44.CrossRefGoogle Scholar
  73. 73.
    Hann DR, Dominguez-Ferreras A, Motyka V, Dobrev PI, Schornack S, Jehle A, et al. The Pseudomonas type III effector HopQ1 activates cytokinin signaling and interferes with plant innate immunity. New Phytol. 2014;201:585–98.CrossRefGoogle Scholar
  74. 74.
    Glazebrook J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol. 2005;43:205–27.CrossRefGoogle Scholar
  75. 75.
    De Vleesschauwer D, van Buyten E, Satoh K, Balidion J, Mauleon R, et al. Brassinosteroids antagonize gibberellin- and salicylate-mediated root immunity in rice. Plant Physiol. 2012;158:1833–46.CrossRefGoogle Scholar
  76. 76.
    Nahar K, Kyndt T, Hause B, Hofte M, Gheysen G. Brassinosteroids suppress rice defense against root-knot nematodes through antagonism with the jasmonate pathway. Mol Plant-Microbe Interact. 2013;26:106–15.CrossRefGoogle Scholar
  77. 77.
    Kazan K, Lyons R. Intervention of phytohormone pathways by pathogen effectors. Plant Cell. 2014;26:2285–09.CrossRefGoogle Scholar
  78. 78.
    Ma K-W, Ma W. Phytohormone pathways as targets of pathogens to facilitate infection. Plant Mol Biol. 2016;91:713–25.CrossRefGoogle Scholar
  79. 79.
    Monaghan J, Zipfel C. Plant pattern recognition receptor complexes at the plasma membrane. Curr Opin Plant Biol. 2012;15(4):349–57.CrossRefGoogle Scholar
  80. 80.
    Trdá L, Boutrot F, Claverie J, Brulé D, Dorey S, Poinssot B. Perception of pathogenic or beneficial bacteria and their evasion of host immunity: pattern recognition receptors in the frontline. Front Plant Sci. 2015;6:219.  https://doi.org/10.3389/fpls.2015.00219.CrossRefPubMedPubMedCentralGoogle Scholar
  81. 81.
    Jones JD, Dangl JL. The plant immune system. Nature. 2006;444(7117):323–9.CrossRefGoogle Scholar
  82. 82.
    Schulze S, Kay S, Büttner D, Egler M, Eschen-Lippold L, Hause G, et al. Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity. New Phytol. 2012;195:894–11.CrossRefGoogle Scholar
  83. 83.
    Jha G, Rajeshwari R, Sonti RV. Functional interplay between two Xanthomonas oryzae pv. oryzae secretion systems in modulating virulence on rice. Mol Plant-Microbe Interact. 2007;20(1):31–40.CrossRefGoogle Scholar

Copyright information

© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Chinese Academy of Agricultural SciencesInstitute of Vegetables and FlowersBeijingChina
  2. 2.Citrus Research and Education Center, Department of Microbiology and Cell ScienceUniversity of FloridaLake AlfredUSA

Personalised recommendations