The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens

Abstract

Riboregulation involving regulatory RNAs, RNA chaperones, and ribonucleases is fundamental for the rapid adaptation of gene expression to changing environmental conditions. The gene coding for the RNase YbeY belongs to the minimal prokaryotic genome set and has a profound impact on physiology in a wide range of bacteria. Here, we show that the Agrobacterium tumefaciensybeY gene is not essential. Deletion of the gene in the plant pathogen reduced growth, motility, and stress tolerance. Most interestingly, YbeY is crucial for A. tumefaciens-mediated T-DNA transfer and tumor formation. Comparative proteomics by using isobaric tags for relative and absolute quantitation (iTRAQ) revealed dysregulation of 59 proteins, many of which have previously been found to be dependent on the RNA chaperone Hfq. YbeY and Hfq have opposing effects on production of these proteins. Accumulation of a 16S rRNA precursor in the ybeY mutant suggests that A. tumefaciens YbeY is involved in rRNA processing. RNA coimmunoprecipitation-sequencing (RIP-Seq) showed binding of YbeY to the region immediately upstream of the 16S rRNA. Purified YbeY is an oligomer with RNase activity. It does not physically interact with Hfq and thus plays a partially overlapping but distinct role in the riboregulatory network of the plant pathogen.IMPORTANCE Although ybeY gene belongs to the universal bacterial core genome, its biological function is incompletely understood. Here, we show that YbeY is critical for fitness and host-microbe interaction in the plant pathogen Agrobacterium tumefaciens Consistent with the reported endoribonuclease activity of YbeY, A. tumefaciens YbeY acts as a RNase involved in maturation of 16S rRNA. This report adds a worldwide plant pathogen and natural genetic engineer of plants to the growing list of bacteria that require the conserved YbeY protein for host-microbe interaction.

Keywords: Agrobacterium tumefaciens; Hfq; RNase; YbeY; iTRAQ; rRNA; ribosome; virulence.

FIG 1

Genetic context and transcription of the A. tumefaciens ybeY. (A) Atu0358 shares high sequence identity with the YbeY orthologues from S. meliloti, V. cholerae, and E. coli. (B) The ybeY gene cluster in A. tumefaciens consists of the phosphate starvation-inducible protein gene phoH (atu0357), ybeY (atu0358), and the hemolysin gene atu0359. (C) Data from the differential RNA-Seq analysis by Wilms’ et al. (42). The transcription start site of ybeY lies upstream of phoH. cDNA libraries were generated from cultures grown without (–Vir) and with (+Vir) virulence induction by acetosyringone. Screenshots of the results after treatment with terminator 5ʹ-phosphate-dependent exonuclease (TEX) to enrich for primary 5ʹ ends are shown.

FIG 2

Growth-phase-dependent production of YbeY^3×Flag. (A and B) The A. tumefaciens YbeY^3×Flag and Hfq^3×Flag strains were cultivated in YEB medium and cells were harvested from the exponential (OD₆₀₀ = 0.5) to the stationary (OD₆₀₀ = 1.5) growth phases. Proteins were separated by SDS-PAGE and detected via a monoclonal anti-Flag M2 antibody after Western blotting. GroEL served as a loading control. (C) Direct comparison of Hfq^Flag (1:5-diluted protein extracts) and YbeY^Flag amounts on one Western blot. (D) WT, Hfq^3×Flag, and YbeY^3×Flag strains were cultivated under noninduced (–Vir) and virulence-induced (+Vir) conditions. Protein amounts were independent of the virulence inducer acetosyringone (AS). The membrane was cut (line), and the T4SS protein VirB9 was detected via a VirB9-specific antibody (upper part) to verify successful virulence induction.

FIG 3

YbeY influences A. tumefaciens growth and motility. (A) Growth experiments with WT, Δhfq, ΔybeY, and Δhfq ΔybeY strains revealed reduced growth of the ybeY mutant compared to the WT. Still, growth deficiency was less severe than in the hfq mutant. Deletion of both genes (Δhfq ΔybeY) led to a severe growth defect. (B) Growth deficiencies of the Δhfq ΔybeY mutant were rescued by ectopic expression of hfq or ybeY from A. tumefaciens. Homologues from E. coli (ybeY^Ec) or S. meliloti (ybeY^Sm) did not complement the growth defect. (C) Swimming of A. tumefaciens WT, Δhfq, and ΔybeY strains on low concentration agar. Swimming distance of Δhfq (***, P = 0.001) and ΔybeY (**, P = 0.009) strains was reduced to about 70% compared to WT. (D) Cultures of WT, Δhfq and ΔybeY strains were grown to exponential phase (OD₆₀₀ = 0.5), and serial dilutions were applied to LB agar plates. WT growth and ΔybeY strain growth were comparable, whereas the growth of the Δhfq strain was reduced ∼10-fold compared to WT growth (upper panel). Addition of 10 μg/ml spectinomycin plus 3 μg/ml streptomycin had the strongest effect on the ybeY deletion strain but also inhibited growth of the hfq mutant (lower panel). EV, empty vector.

FIG 4

YbeY is crucial for A. tumefaciens virulence. (A) Infection of potato tuber discs with 10⁶ cells of WT, Δhfq, and ΔybeY strains. After incubation for 6 weeks (16-h light/dark cycle, 23°C), tumors were counted per disc, and the average of 100 infected potato discs per strain was calculated. WT infection led to an average of seven to eight tumors per disc. The infection efficiency of Δhfq (one to two tumors/disc) and ΔybeY (two to three tumors/disc) strains was drastically reduced (****, P < 0.0001). (B) Similar results were obtained from A. thaliana root infection assays. About 25% of the 300 roots were infected by either the hfq (**, P = 0.003) or the ybeY (*, P = 0.015) mutant in contrast to the ∼55% infection efficiency of A. tumefaciens WT. (C) Qualitative measurement of T-DNA transfer was performed using WT, Δhfq, and ΔybeY strains harboring a plasmid-carried GUS-reporter gene system (pBISN1). Successful transfer of T-DNA (gus) resulted in β-glucuronidase expression in the infected plant tissue. Cleavage of the substrate X-Glc by the β‑glucuronidase stained the corresponding plant tissue (blue). Infection with WT^pBISN1 led to extensive staining of the leaves, whereas seedling infection by hfq^pBISN1 or ybeY^pBISN1 mutants was less efficient.

FIG 5

YbeY affects vir gene expression and Vir protein amounts. (A) WT, Δhfq, and ΔybeY strains harboring a plasmid-carried virB-lacZ fusion were cultured under virulence inducing (+Vir) and noninducing conditions (–Vir). Under +Vir conditions, the β-galactosidase activity was reduced in the ybeY mutant, whereas deletion of hfq did not affect virB-lacZ expression significantly compared to WT activity. (B) Protein amounts of VirB2 (12.3 kDa), VirB5 (23.3 kDa), VirB8 (26.3 kDa), and VirB9 (32.2 kDa) under the conditions described for panel A were detected via Western blotting. Vir proteins were detectable only under +Vir conditions. Vir protein amounts were slightly increased in the Δhfq strain but did not show significant differences in the ΔybeY strain. GroEL (57.4 kDa) served as a loading control.

FIG 6

Global proteome analysis of A. tumefaciens WT and ΔybeY strains. Quantification of the WT and ΔybeY proteomes was performed using isobaric tags for relative and absolute quantitation (iTRAQ) and LC-MS/MS. (A) A total of 2,544 proteins were identified in three biological replicates. Based on a confidence interval of 95%, 26 proteins were upregulated (ΔybeY/WT ratio of >1.35) and 33 proteins were downregulated (ΔybeY/WT ratio of <0.75) in a ybeY deletion strain. (B) Proteins regulated by YbeY were clustered according to KEGG orthology enrichment into transporters, enzymes, transcription, motility and chemotaxis, translation, diverse cellular functions, and proteins with unknown function. (C) Overlap between Hfq- and YbeY-dependent proteins. Twelve YbeY-dependent proteins were previously identified to be influenced by Hfq (41).

FIG 7

YbeY participates in rRNA processing. (A) Deletion of ybeY resulted in an additional rRNA fragment (17S rRNA). The fragment was most prominent in the exponential growth phase. Complementation (+ybeY) of the ΔybeY strain resulted in WT-like rRNA profile. (B) RIP-Seq experiments revealed binding of YbeY^3×Flag to the 5ʹ upstream region of the 16S rRNA (upper panel). The 17S rRNA was isolated from an agarose gel, and the 5ʹ end was identified via 5ʹ RACE (lower panel). Sequencing of the RACE PCR fragment revealed extension of the 16S rRNA by 173 nucleotides in the ybeY mutant.

FIG 8

Purification and biochemical analyses of A. tumefaciens. (A) GPC with YbeY^6×His (20.6 kDa, C-terminally tagged) resulted in a distinct protein peak with a maximum absorption of 719.5 mAU at 14.3 ml, indicating elution of 34- to 86-kDa large protein complexes, which corresponded to homo-oligomers of two to four YbeY^6×His monomers. Protein abundance in fractions B to G was verified by SDS-PAGE. (B) RNase activity of purified YbeY. Various amounts of N-terminally tagged YbeY^6×His protein (50 to 250 μM) were incubated with 5 μg total RNA from A. tumefaciens WT. Lane B, 100 μM BSA; lane R, 35 μM RNase A. (C) Pulldown experiments of YbeY^6×His and Hfq^3×Flag protein variants from cell extracts after incubation for 30 min on ice. YbeY^6×His was bound by the Ni-NTA column, whereas Hfq^3×Flag was washed off the column by low imidazole concentrations (5 to 10 mM). YbeY^6×His was eluted with 250 mM imidazole. Western blot analysis with specific His- or Flag-tagged antibodies did not show coelution of Hfq^3×Flag protein with YbeY^6×His. Similar results were obtained when protein tags were switched between both proteins (YbeY^3×Flag and Hfq^6×His).