Small non-coding RNAs in plant-pathogenic Xanthomonas spp

Abstract

The genus Xanthomonas comprises a large group of plant-pathogenic bacteria. The infection and bacterial multiplication in the plant tissue depends on the type III secretion system and other virulence determinants. Recent studies revealed that bacterial virulence is also controlled at the post-transcriptional level by small non-coding RNAs (sRNAs). In this review, we highlight our current knowledge about sRNAs and RNA-binding proteins in Xanthomonas species.

Keywords: :

• transcriptome
• Xanthomonas
• plant pathogens
• sRNAs
• RNA-binding proteins
• post-transcriptional regulation
• bacteria

Introduction

Gram-negative γ-proteobacteria of the genus Xanthomonas are economically important pathogens of many plant species, including crop plants, and are grouped into pathovars (pv.) according to their host range.¹ During infection, the bacteria enter the plant tissue via natural openings like stomata and hydathodes or wounds. The bacteria multiply either locally in the intercellular spaces, as in case of Xanthomonas campestris pv. vesicatoria (Xcv), X. oryzae pv. oryzicola (Xoc), and X. axonopodis pv. citri (Xac), or spread systemically via the plant vascular system, e.g., X. campestris pv. campestris (Xcc) and X. oryzae pv. oryzae (Xoo).² While Xanthomonas spp. only infect plants, another member of the Xanthomonadaceae family, Stenotrophomonas maltophilia (Smal), is an opportunistic human pathogen.³

Pathogenicity of Xanthomonas spp. relies on a type III secretion system (T3SS), which is encoded by the chromosomal hrp (hypersensitive response and pathogenicity)-gene cluster.⁴ The T3SS is induced during infection and translocates bacterial effector proteins (T3Es) directly into the plant cell.² In addition, other protein secretion systems contribute to virulence of Xanthomonas spp.⁵

In our laboratory, we study the sequenced model pathogen Xcv and its interaction with pepper and tomato plants.⁶ Multiplication of Xcv in susceptible host plants causes bacterial spot symptoms, that later become necrotic. By contrast, resistant host cultivars recognize a certain bacterial T3E, which often induces the hypersensitive response (HR). The HR is a rapid local plant cell death, accompanied by the arrest of bacterial multiplication.⁷

In the last two decades, bacterial non-coding RNAs (ncRNAs) have emerged as important post-transcriptional regulators, because they affect mRNA stability and/or translation and the activity of proteins.⁸ ncRNAs encompass trans-encoded small RNAs (sRNAs), protein-binding sRNAs, cis-encoded antisense RNAs, and cis-regulatory mRNA elements, i.e., riboswitches.⁸ In many bacteria, sRNAs were shown to contribute to environmental adaptation, e.g., carbon utilization and the bacterial response to stress.⁹ In addition, sRNAs in animal- and human-pathogenic bacteria are known to affect quorum sensing and the expression of virulence genes,^10,11 e.g., RNAIII in Staphylococcus aureus and sRNAs of the Qrr family in Vibrio cholerae.^12-14 To date, the knowledge of sRNAs in plant-pathogenic bacteria is limited to Agrobacterium tumefaciens, Pseudomonas syringae pv. tomato, and members of the genera Erwinia and Xanthomonas.^15-24

This review focuses on sRNAs and the RNA-binding proteins (RBPs) Hfq and CsrA/RsmA in Xanthomonas spp. We will only discuss sRNAs, which have been validated by northern blot analyses and apologize to all colleagues whose data were not discussed in detail for space reasons.

Identification of sRNAs in Xanthomonas spp.

In members of the genus Xanthomonas, sRNAs were so far identified in Xcv, Xcc, and Xoo.^19-23 The next paragraphs are subdivided accordingly.

Xcv—Differential RNA-Seq Analysis Revealed 24 sRNAs

In 2012 we provided a first insight into the transcriptome of Xcv strain 85-10 using a differential RNA-seq (dRNA-seq) approach, which permits the discrimination of primary and processed transcripts, and thus, enables the identification of transcription start sites (TSSs).²² In contrast to previous studies of other bacterial species, which were based on the tedious manual inspection of dRNA–seq data, we applied an automated statistical approach and mapped 1421 TSSs in Xcv.²² Eight hundred and thirty-one of these presumably represent the TSSs of 17.35% of all annotated coding sequences (CDSs). Interestingly, 14% of the identified mRNAs lack a 5′-untranslated region (UTR), whereas 13% carry unusually long 5′-UTRs ranging from 150–300 bp, including six mRNAs that encode T3Es (xopAA, xopB, avrBs1, xopC, xopD, and xopN).²² The long UTRs might harbor cis-regulatory elements, such as riboswitches, or serve as binding sites for post-transcriptional regulators, i.e., sRNAs or proteins. Notably, the CDSs of some effector genes might be larger than annotated in the genome, as recently demonstrated for xopD.^6,25 The dRNA–seq analysis also revealed a plethora of antisense RNAs in Xcv, some of which are associated with virulence genes that encode components (HrcC) or substrates of the T3SS (AvrBs1, XopAA, XopB, XopD, XopE2, and XopO).²² It is tempting to speculate that these antisense RNAs could prevent the intracellular accumulation of potentially cytotoxic T3Es under conditions that induce the hrp–regulon, but do not allow for T3S, as in case of Xcv grown in XVM2 medium.²⁶ However, this hypothesis remains to be tested. Overall, the dRNA–seq study revealed that 22% of all nucleotides, which belong to annotated CDSs, are covered by antisense reads.²² It has been hypothesized that antisense transcription over a great portion of the genome might play a role in fine-tuning of gene expression.²⁷

The major aim of the dRNA–seq analysis of Xcv was the identification of sRNAs. In total, 23 potential regulatory RNAs and 6S RNA were confirmed by northern blot analyses, 15 of which represent sRNAs (sX1 to 15) and eight represent cis-encoded antisense RNAs (asX1 to 7 and PtaRNA1; Table 1).^19,22 Except for sX6, which was shown to encode a small protein, the identified RNAs are non-coding. Bioinformatic analyses suggest that one of the abundant antisense RNAs, PtaRNA1 (plasmid-transferred antisense RNA1), represents a chromosomally encoded RNA-regulator of a novel toxin–antitoxin locus.¹⁹ Such type I toxin–antitoxin loci employ an antisense RNA to post-transcriptionally repress the synthesis of toxic proteins.²⁸ However, the regulatory function of PtaRNA1 and the presumed toxic character of the potential target mRNA (XCV2162) awaits further analyses. The Xcv transcriptome analysis also provided evidence for a role of sRNAs in virulence, because the abundance of five sRNAs, including sX12, and three antisense RNAs was affected by the key regulators of the T3SS, HrpG, and HrpX.²² Further analysis revealed that sX12 promotes Xcv virulence, but does not affect in planta growth and T3S in vitro.²² So far, potential targets of sX12 are unknown and the functional mechanism needs to be explored. To date, the Xcv sRNA sX13 is the most extensively studied sRNA in Xanthomonas spp. with respect to potential targets and physiological functions and is discussed below in detail. Altogether, the dRNA–seq approach in Xcv provided a first comprehensive insight into the transcriptome of a Xanthomonas strain and significantly contributed to the improvement of the genome annotation.^6,22

Table 1. Rfam classification of verified sRNAs from Xanthomonadaceae.

Accession^a	ID	Members^b	Features	Ref.
RF02222	sX2	sX2	-	^22
RF02223	sX4	sX4	HrpG/HrpX-dependent accumulation	^22
RF02224	sX5	sX5	HrpX-induced	^22
		SmsR26	Hfq-dependent accumulation	^34
RF02225	sX6	sX6	-	^22
RF02226	sX7	sX7	-	^22
RF02227	sX8	sX8	HrpX-induced	^22
RF02228	sX9	sX9	-	^22
RF02230	sX11	sX11	HrpG/HrpX-repressed	^22
		SmsR35	-	^34
RF02231	sX12	sX12	HrpX-induced, contributes to virulence	^22
RF02232	sX13	sX13	Regulator of virulence, Hfq-independent accumulation	^22 ^, ^24
		SmsR39	Hfq-independent accumulation	^34
RF02233	sX14	sX14	Hfq-dependent accumulation	^22 ^, ^24
		sRNA-Xoo3	Hfq-dependent accumulation	^21
RF02234	sX15	sX15	-	^22
RF02235	asX1	asX1	HrpG/HrpX-dependent accumulation	^22
RF02236	asX2	asX2	-	^22
RF02237	asX3	asX3	-	^22
RF02238	asX4	asX4	HrpX-induced	^22
		sRNA-Xoo4	Hfq-independent accumulation	^21
RF02239	asX6	asX6	-	^22
RF02221	sRNA-Xcc1	sRNA-Xcc1	HrpX-induced	^20 ^, ^54
		sX1	-	^22
		sX10	-	^22
		sRNA-Xoo6	Hfq-independent accumulation	^21
		SmsR6	-	^34
RF02240	sRNA-Xoo1	sRNA-Xoo1	Hfq-dependent accumulation	^21
		SmsR36	Hfq-dependent accumulation	^34
RF02241	sRNA-Xoo2	sRNA-Xoo2	Hfq-dependent accumulation	^21
RF02242	sRNA-Xoo5	sRNA-Xoo5	Hfq-independent accumulation	^21
RF02243	sRNA-Xoo8	sRNA-Xoo8	Hfq-independent accumulation	^21
RF01811	PtaRNA1	PtaRNA1	Predicted type I antitoxin	^19

a Rfam release Rfam 11.0 (August 2012).^{29 b} sRNAs verified by northern blot in: Xcv 85-10 (sX/asX), Xcc 8004 (sRNA-Xcc), Xoo PXO99 (sRNA-Xoo), and Smal K279a (SmsR).

Xcc—Shotgun Cloning and RNA–Seq Revealed 12 sRNAs

In 2010 Jiang et al. reported the identification of four sRNAs in Xcc strain 8004 using shotgun cloning and sequencing of cDNA derived from size-selected RNA (50–500 nt).²⁰ The respective sRNA candidates were validated by northern blot and termed sRNA-Xcc1 (Table 1) to 4. Although potential mRNA-targets of the Xcc sRNAs were predicted,²⁰ the presumed base-pairing function of these transcripts is questionable, since Rfam searches (Rfam 11.0; Aug. 2012)²⁹ suggest that sRNA-Xcc2, -Xcc3, and -Xcc4 represent orthologs of the conserved 6S RNA, SRP RNA, and 5S rRNA.

In another study, an RNA–seq approach of Xcc strain 8004 was used to identify genes, whose expression is affected by mutation of rpf (regulation of pathogenicity factors)-genes.²³ The Rpf-regulatory system in Xcc controls the production and perception of the quorum-sensing autoinducer DSF (diffusible signal factor) and contributes to biofilm formation and the production of secreted virulence factors, i.e., extracellular enzymes and the extracellular polysaccharide xanthan.^30,31 The RNA–seq study suggests the existence of previously not-annotated genes, including about 150 potential virulence factors and 24 sRNA candidates.²³ Considering that the approach did not allow strand-specific assignment of sequencing reads, it is possible that some of the sRNA candidates are derived from processed mRNAs or represent cis-encoded antisense RNAs. Eight sRNAs were validated by northern blot, which showed that the expression of three sRNAs (sRNA-Xcc15, -Xcc16, and -Xcc28), was Rpf-dependent. Intriguingly, the virulence of an Xcc triple mutant deleted in the three sRNA genes was reduced compared with the wild-type, whereas virulence of single mutants was not altered.²³

Xoo—Shotgun Cloning Identified Eight sRNAs

In 2011 Liang et al. described the identification of eight sRNAs in Xoo strain PXO99 based on computational prediction, shotgun cloning, and sequencing of cDNA derived from size-selected RNA (50–500 nt).²¹ The expression of these sRNAs, termed sRNA-Xoo1 to 8, was verified by northern blot. Xoo single mutants deleted in each of the seven sRNA genes (sRNA-Xoo1 to 7) did not exhibit phenotypic changes compared with the wild-type with respect to in vitro growth, secretion of extracellular enzymes, and virulence.²¹ Proteome analyses suggest that sRNA–Xoo1 might be involved in the regulation of amino acid metabolism and transport.²¹ Such a role was also demonstrated for the Salmonella sRNA GcvB.^32,33 In addition, the proteome data imply a role of sRNA–Xoo3 in the regulation of oxidation-reduction processes, stress response, and metabolic pathways.²¹ A role in housekeeping processes was suggested for sRNA–Xoo4, which affects the abundance of proteins involved in DNA replication, rRNA processing, and metabolism.²¹ It will be interesting to determine whether the impact of Xoo sRNAs on protein abundance is due to base-pairing interactions with corresponding mRNAs.

Phylogenetic Distribution of sRNAs in Xanthomonas spp.

At present, the Rfam database (Rfam 11.0; updated Aug. 2012)²⁹ comprises 23 sRNA families, whose founding member is a Xanthomonas sRNA (Table 1). Many of the corresponding sRNAs were first described in Xcv.^19,22 Phylogenetic analyses suggest that the majority of Xanthomonas sRNAs is exclusively conserved in members of the Xanthomonadaceae family. Exceptions include PtaRNA1 from Xcv, which exhibits an erratic phylogenetic distribution, and widely conserved sRNAs with housekeeping functions, e. g. 6S RNA, SRP RNA, tmRNA, and RNase P.^19,22,34 Homologs of some of the clade-specific sRNAs were identified by independent studies in different Xanthomonas spp. and in Smal (Table 1). While similar lengths were reported for homologs like sX13 (115 nt) and SmsR39 (113 nt), the reported lengths of asX4 (309 nt) and sRNA-Xoo4 (145 nt) differ significantly. The Xcv sRNA sX13 is highly conserved in members of the Xanthomonadaceae (Fig. 1A) and originates from a locus downstream of the DNA–polymerase I-encoding polA gene.²⁴ Most interestingly, this locus encodes the sX13-unrelated Spot42 sRNA in Escherichia coli and members of the αr7 sRNA family in α-proteobacteria.^35,36 Thus, the polA locus appears to be a hotspot for sRNA genes in distantly related bacteria.

Figure 1. (A) Conservation of sX13. LocARNA prediction of the consensus secondary structure of all sX13 family members in xanthomonads listed in the Rfam database (Rfam 11.0, Aug. 2012).^29,55 Compensatory mutations that retain the sX13 structure are indicated by circles. (B) Model of potential physiological functions of sX13 in Xcv according to Schmidtke et al.²⁴ sX13 accumulates in response to extracellular stimuli and indirectly promotes HrpG activation, which is induced by unknown signaling pathways.^56,57 sX13 supposedly impacts on motility, quorum sensing (QS), and the activity of other sRNAs by repressing Hfq synthesis. Boxed arrows, wavy lines, and circles indicate genes, mRNAs, and proteins. Dashed circles mark unknown proteins or signaling pathways. Green, red, and dashed arrows denote positive, negative, and putative effects.

sX13 from Xcv Regulates Virulence

While the biological functions of most sRNAs identified in Xanthomonas spp. are unknown, a role in the pathogen–plant interaction was reported for sRNAs from Xcc and Xcv (Table 1).^22-24 However, insights into underlying regulatory mechanisms are limited to sX13, which we recently discovered as novel regulator of virulence gene expression in Xcv strain 85–10.²⁴ Deletion of sX13 affected bacterial growth in complex and minimal medium and severely reduced Xcv virulence. This was unexpected, because sX13 is constitutively expressed, whereas most known Xcv virulence factors are co-expressed with the T3SS, e. g., T3Es and sRNA sX12.^2,22,24 While sX13 is not part of the hrp-regulon, it promotes mRNA accumulation of hrpX and HrpX-induced genes, which encode components and substrates of the T3SS. By contrast, the mRNA level of hrpG, the master regulator of the T3SS, was not affected. Complementation studies suggest that sX13 acts upstream of HrpG and indirectly affects HrpG activity, presumably by targeting an upstream regulator.²⁴ Although post-translational control of HrpG activity was recently reported for Xcc,³⁷ signals or proteins that activate HrpG in Xcv are unknown.

Potential functions of sX13 extend beyond the regulation of the T3SS, as implied by microarray analyses.²⁴ The sX13 regulon comprises more than 60 genes involved in virulence, quorum sensing, and motility. This suggests that sX13 coordinates the activity of different regulatory networks in response to environmental changes (Fig. 1B). Such a role is highlighted further by the increased accumulation of sX13 under certain stress conditions.²⁴ Because the microarray analyses did not allow for the identification of mRNAs that are directly targeted by sX13, some genes are probably indirectly affected on the transcriptional level. Intriguingly, sX13 might also impact on post-transcriptional processes, because it represses the accumulation of the hfq mRNA and synthesis of Hfq (Fig. 1B),²⁴ which encodes a conserved RBP required for sRNA activity in most bacteria (see below).

Overall, there is striking evidence that sX13 acts on certain mRNAs via antisense base-pairing: the sX13 structure consists of three stems and apical C-rich loops (Fig. 1A), whereas 70% of the negatively regulated mRNAs carry complementary G-rich motifs in proximity of the translation start codon.²⁴ Complementation studies demonstrated that both C-rich sX13 loops and G-rich mRNA motifs are required for sX13-mediated repression of target–mRNA expression, suggesting base-pairing interactions. However, complementary mutations in sX13 loops and the G-rich mRNA motif did not restore sX13-dependency, which might be due to the fact that mutation of G-rich motifs decreased or abolished mRNA translation. Thus, potential sX13-targeting sites in mRNAs appear to serve as translational enhancers, implying that sX13 interferes with enhancer activity.²⁴ A similar case was reported for the Salmonella sRNA GcvB, which targets “CA”-rich enhancer elements.^32,33

Despite the obvious redundancy, the C-rich sX13 loops differentially contribute to sX13 activity: while loops 2 and 3 promote Xcv virulence, only loop 2 appears to have a major role in target–mRNA repression.²⁴ So far, potential targets of loop 1 and loop 3 are elusive. It will be interesting to see whether multiple sX13 loops contribute to the regulation of some of the presumed target–mRNAs, which contain multiple G-rich motifs. Such a mechanism is employed by RNAIII of the Gram-positive pathogen S. aureus,¹⁴ which resembles sX13 with respect to C-rich loops. Besides structural similarity of sX13 and RNAIII, the latter also regulates the expression of virulence genes.^14,38

The RNA Chaperone Hfq

Hfq is a widely conserved bacterial RBP, which has been extensively studied in enterobacteria and in most cases facilitates base-pairing interactions of sRNAs and their target-mRNAs.^39,40 Furthermore, Hfq is assumed to protect sRNAs from degradation, as the stability of Hfq-dependent sRNAs decreases in absence of Hfq. Inactivation of Hfq often results in pleiotropic phenotypes, e. g., reduced bacterial growth, altered motility, and stress responses, as well as decreased virulence of pathogenic bacteria.^11,41

In Xanthomonas spp., the role of Hfq was investigated in Xoo and Xcv using in-frame deletion mutagenesis and a frame-shift mutation, respectively. Hfq contributes to growth of Xoo in complex medium, but does not appear to play a role in virulence.²¹ Similarly, inactivation of hfq in Xcv did not affect virulence, which was surprising, because the sRNA sX13 contributes to plant infection and represses Hfq synthesis.²⁴ Thus, our findings demonstrate that sX13 acts Hfq-independently, which is supported by the fact that sX13-dependent expression of putative target mRNAs is not affected in the hfq mutant.²⁴ It remains to be seen whether yet-unidentified RBPs in Xanthomonas are required for the activity of sX13 and other sRNAs. Similar to sX13 in Xcv, the S. aureus RNAIII controls virulence gene expression in an Hfq-independent manner.⁴² In other plant–pathogenic bacteria, i.e., A. tumefaciens and Erwinia spp., Hfq was shown to contribute to virulence.^43,44 In addition, Hfq is involved in symbiotic interactions of S. meliloti with the plant.⁴⁵

Although the function of Hfq is obscure in Xanthomonas spp., the presumed role as RBP is supported by the Hfq-dependent accumulation of the Xcv sRNA sX14 and three Xoo sRNAs, including the sX14 homolog sRNA–Xoo3 (Table 1).^21,24 In Smal, two Hfq-dependent sRNAs were identified, one of which, SmsR36,³⁴ is homologous to the Hfq-dependent sRNA–Xoo1 (Table 1). Hfq-independent Smal sRNAs include SmsR39, which is homologous to sX13 from Xcv. Overall, Hfq appears to have different roles in Xanthomonas spp. and the related Smal, as an hfq mutation in Smal impacts on growth, motility, biofilm formation, tolerance against antimicrobial agents, and the bacterial interaction with eukaryotic cells.³⁴ Further research is needed to address physiological functions and the RNA-binding capability of Hfq in Xanthomonas spp.

The CsrA-/RsmA-Regulatory System

Besides Hfq, proteins of the CsrA/RsmA family are well-studied bacterial RBPs, which contribute to a variety of cellular processes e.g., glycogen biosynthesis, flagellum-mediated motility, biofilm formation, and virulence.^46,47 Members of this RBP family bind to the 5′-UTRs of target mRNAs, and thus, repress translation and stimulate mRNA decay or, in a few cases, act positively on target mRNAs.⁴⁸ In E. coli, the activity of CsrA is modulated by binding of the sRNAs CsrB and CsrC, which mimic target mRNAs.^46,49 Similarly, in P. fluorescens, the RsmA activity is controlled by corresponding sRNAs, i. e., RsmX, RsmY, and RsmZ.⁵⁰

In Xanthomonas spp., the role of RsmA has been studied in Xcc and Xoo. In both strains, deletion of rsmA led to a loss of pathogenicity, which was associated with an altered expression of hrp- and rpf-genes.^51-53 These mutants also showed pleiotropic phenotypes, e.g., higher levels of intracellular glycogen, increased bacterial adhesion and aggregation, and reduced production of extracellular enzymes.^51-53 RsmA in Xanthomonas spp. appears to control quorum sensing and pathogenicity. The underlying regulatory mechanism is still elusive, because Xanthomonas spp. lack homologs of the known CsrA/RsmA-binding sRNAs of other bacteria.

Conclusions

Since the discovery of the first sRNAs in Xanthomonas spp. four years ago, 44 sRNAs were experimentally validated in members of the genus, which was substantially fostered by high-throughput transcriptome-sequencing approaches. Since these methods generate huge data sets and reveal many sRNA candidates, careful evaluation by independent methods is needed to confirm sRNA expression, i.e., northern blots. Xcv transcriptomics demonstrated the benefits of dRNA–seq for the improvement of genome annotations and the identification of TSSs and laid an excellent foundation for more in-depth studies. Besides sX13 in Xcv, a role in virulence was reported for other Xanthomonas sRNAs, although their precise function and targets remain to be identified. Future studies will lead to exciting new insights into post-transcriptional regulation of gene expression and virulence of bacterial plant pathogens.

Abbreviations:
bp	=	base pair
CDS	=	coding sequence
dRNA-seq	=	differential RNA-seq
HR	=	hypersensitive response
hrp	=	hypersensitive response and pathogenicity
nt	=	nucleotide
ncRNA	=	non-coding RNA
pv.	=	pathovar
RBP	=	RNA-binding protein
Smal	=	Stenotrophomonas maltophilia
sRNA	=	small RNA
T3E	=	type III effector protein
T3SS	=	type III secretion system
TSS	=	transcription start site
UTR	=	untranslated region
Xac	=	Xanthomonas axonopodis pv. citri
Xcc	=	Xanthomonas campestris pv. campestris
Xcv	=	Xanthomonas campestris pv. vesicatoria
Xoc	=	Xanthomonas oryzae pv. oryzicola
Xoo	=	Xanthomonas oryzae pv. oryzae

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG, Priority program “Sensory and Regulatory RNAs in Prokaryotes,” SPP 1258) to U. Bonas.

10.4161/rna.28240