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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 WTpBISN1 led to extensive staining of the leaves, whereas seedling infection by hfqpBISN1 or ybeYpBISN1 mutants was less efficient.
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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 Agrob...
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... A functional role of RNases in the stress response of numerous microorganisms has been reported already in the past. Often a reduction or lack of the native RNase activity leads to enhanced susceptibility towards various cellular stresses, like oxidative, osmotic or temperature stress (Duggal et al., 2020;Förstner et al., 2018;Lejars & Hajnsdorf, 2022;Möller et al., 2019;. In contrast, our data ( Figure 9) document a higher resistance of R. sphaeroides against oxidative stress when RNase III activity is lacking. ...
RNase III is a dsRNA‐specific endoribonuclease, highly conserved in bacteria and eukarya. In this study, we analysed the effects of inactivation of RNase III on the transcriptome and the phenotype of the facultative phototrophic α‐proteobacterium Rhodobacter sphaeroides. RNA‐seq revealed an unexpectedly high amount of genes with increased expression located directly downstream to the rRNA operons. Chromosomal insertion of additional transcription terminators restored wild type‐like expression of the downstream genes, indicating that RNase III may modulate the rRNA transcription termination in R. sphaeroides. Furthermore, we identified RNase III as a major regulator of quorum‐sensing autoinducer synthesis in R. sphaeroides. It negatively controls the expression of the autoinducer synthase CerI by reducing cerI mRNA stability. In addition, RNase III inactivation caused altered resistance against oxidative stress and impaired formation of photosynthetically active pigment‐protein complexes. We also observed an increase in the CcsR small RNAs that were previously shown to promote resistance to oxidative stress. Taken together, our data present interesting insights into RNase III‐mediated regulation and expand the knowledge on the function of this important enzyme in bacteria.
... Various applications of AGROBEST have been demonstrated in Arabidopsis seedlings, including gusA-intron reporter gene expression (Fig. 2), fluorescent protein localization [19][20][21], bimolecular fluorescence complementation (BiFC) [22,23], co-immunoprecipitation (Co-IP) [24,25], effects of transcription factor actions [26,27], circadian clock monitoring [22], and agroinfiltration for virus inoculation [28]. Besides, AGROBEST is also a convenient method to measure T-DNA transfer efficiency [29][30][31][32][33][34]. Therefore, the AGROBEST method is a fast, simple, reliable, and versatile tool that can be applied for versatile studies. ...
Agrobacterium-mediated transient transformation for gene expression is a simple and fast method to analyze transgene functions in plants. Agroinfiltration in leaves of Nicotiana benthamiana is a common method for transient expression. However, agroinfiltration in leaves of Arabidopsis thaliana is challenging due to the low and variable efficiency. Here, we describe procedures of a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation) for gene expression in A. thaliana seedlings. High efficiency of AGROBEST has been achieved by virulence (vir) gene pre-induction of a specific disarmed Agrobacterium tumefaciens strain C58C1(pTiB6S3ΔT)H followed by co-cultivation with Arabidopsis seedlings in an optimized medium with AB salts and buffered acidic plant culture medium. The stable acidic medium largely increases Agrobacterium-mediated transient expression levels and reduces plant defense responses, suggesting that AGROBEST enables high transient expression efficiency by compromising plant immunity. In summary, AGROBEST is a simple, fast, reliable, and robust transient expression system offering a quick and convenient method to observe protein localization, protein–protein interactions, promoter activities, and gene functional studies in Arabidopsis seedlings.Key words
Arabidopsis
Seedling
Agrobacterium
Transient transformationGene expressionAGROBEST
... Being a 'red button' of the cellular protein synthesis machinery, YbeY appears to 'regulate' the expression of hundreds of genes at both RNA and protein levels [19,[29][30][31][32][33]. This influence occurs via both direct and The primary driver of the pleiotropic phenotypes appears to be a severe small subunit (SSU) biogenesis defect, which manifests itself in the impairment of 16S rRNA 3 0 -processing (in bacteria and chloroplasts) or SSU assembly problems (in mitochondria). ...
... The majority of observed gene expression changes, however, appear to be indirect. Indeed, while the interactions of YbeY with ribosomal components are extensively characterised across various species (see below), there is little evidence of its direct association with protein-coding or regulatory RNAs [33,34]. Instead, a few studies exposed amazingly complex and meandering regulatory cascades underlying some of ΔybeY phenotypes [19,32,35]. ...
... Pervasive gene expression changes often arise when regulators, such as transcription factors or sRNAs, get involved. This is exactly what has been observed in various bacterial ybeY mutants [29,30,32,33,36]. In Pseudomonas aeruginosa, YbeY loss curtails the production of the key σ-factor RpoS, which is responsible for the expression of virulence and stress response genes [19]. ...
YbeY is an ultraconserved small protein belonging to the unique heritage shared by most existing bacteria and eukaryotic organelles of bacterial origin, mitochondria and chloroplasts. Studied in more than a dozen of evolutionarily distant species, YbeY is invariably critical for cellular physiology. However, the exact mechanisms by which it exerts such penetrating influence are not completely understood. In this review, we attempt a transversal analysis of the current knowledge about YbeY, based on genetic, structural, and biochemical data from a wide variety of models. We propose that YbeY, in association with the ribosomal protein uS11 and the assembly GTPase Era, plays a critical role in the biogenesis of the small ribosomal subunit, and more specifically its platform region, in diverse genetic systems of bacterial type.
... In E. coli, YbeY has been shown to play important roles in bacterial resistance to heat shock, oxidative stresses, and a variety of antibiotics (33). Deletion of the ybeY gene in the plant pathogen Agrobacterium tumefaciens reduced the bacterial growth rate, motility, and stress tolerance (34). In Yersinia enterocolitica serotype O:3, YbeY is involved in the regulation of the genes of the Yersinia virulence plasmid (pYV) and multiple regulatory small RNAs (11). ...
YbeY is a highly conserved RNase in bacteria and plays essential roles in the maturation of 16S rRNA, regulation of small RNAs (sRNAs) and bacterial responses to environmental stresses. Previously, we verified the role of YbeY in rRNA processing and ribosome maturation in Pseudomonas aeruginosa and demonstrated YbeY-mediated regulation of rpoS through a sRNA ReaL. In this study, we demonstrate that mutation of the ybeY gene results in upregulation of the type III secretion system (T3SS) genes as well as downregulation of the type VI secretion system (T6SS) genes and reduction of biofilm formation. By examining the expression of the known sRNAs in P. aeruginosa , we found that mutation of the ybeY gene leads to downregulation of the small RNAs RsmY/Z that control the T3SS, the T6SS and biofilm formation. Further studies revealed that the reduced levels of RsmY/Z are due to upregulation of retS . Taken together, our results reveal the pleiotropic functions of YbeY and provide detailed mechanisms of YbeY-mediated regulation in P. aeruginosa .
IMPORTANCE Pseudomonas aeruginosa causes a variety of acute and chronic infections in humans. The type III secretion system (T3SS) plays an important role in acute infection and the type VI secretion system (T6SS) and biofilm formation are associated with chronic infections. Understanding of the mechanisms that control the virulence determinants involved in acute and chronic infections will provide clues for the development of effective treatment strategies. Our results reveal a novel RNase mediated regulation on the T3SS, T6SS and biofilm formation in P. aeruginosa .
... One such factor is the UPF0054 family protein YbeY found in all classified bacteria (2). Based on studies in various bacteria, YbeY has been implicated in ribosome maturation and quality control, with a particularly important role in small subunit (SSU) biogenesis (3)(4)(5)(6)(7)(8), and post-transcriptional gene expression regulation (9)(10)(11)(12)(13)(14). The deletion of ybeY is often lethal or associated with severe alterations of cellular metabolism and growth, indicating its indispensability for a wide variety of bacterial-type ribosomes (4,6,7,(13)(14)(15)(16)(17). ...
... Based on studies in various bacteria, YbeY has been implicated in ribosome maturation and quality control, with a particularly important role in small subunit (SSU) biogenesis (3)(4)(5)(6)(7)(8), and post-transcriptional gene expression regulation (9)(10)(11)(12)(13)(14). The deletion of ybeY is often lethal or associated with severe alterations of cellular metabolism and growth, indicating its indispensability for a wide variety of bacterial-type ribosomes (4,6,7,(13)(14)(15)(16)(17). ...
... The deeply conserved YbeY protein has recently come into limelight as a critical factor required for normal physiol-ogy in Bacteria. Its loss has been associated with a wide array of debilitating phenotypes, such as sensitivity to abiotic stresses, inability to establish host-pathogen/symbiont relationships (6,7,(13)(14)(15), metabolic deregulations and a severe growth impairment up to lethality (3,11,17). Widespread among Eukarya, YbeY has so far only been studied in A. thaliana, where it was found indispensable for chloroplast development and photosynthesis (16). ...
Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.
... The deeply conserved YbeY protein has recently come into limelight as a critical factor required for normal physiology in Bacteria. Its loss has been associated with a wide array of debilitating phenotypes, such as sensitivity to abiotic stresses, inability to establish host-pathogen/symbiont relationships (6,7,(13)(14)(15), metabolic deregulations and a severe growth impairment up to lethality (3,11,17). Widespread among Eukarya, YbeY has so far only been studied in A. thaliana, where it was found indispensable for chloroplast development and photosynthesis (16). ...
Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3’-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter recruits uS11m to the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.
... In Corynebacterium glutamicum, YbeY is implicated in the maturation of 4.5S rRNA (25). In Agrobacterium tumeficiens, loss of YbeY has been shown to result in accumulation of 16S rRNA precursors (26), while in Brucella abortus loss of YbeY resulted in sensitivities to various stresses and significantly reduced the ability to grow in macrophages (27). ybeY is also identified to be one of the genes required for longevity under carbon starvation conditions in Rhodopseudomonas palustris, a phototrophic alpha-proteobacterium (28). ...
... Total RNA profile shows that the EcΔybeY pWSK29 strain accumulates 17S rRNA precursor and the 16S* band ( Figure 8A). The 16S* is a 16S degradation band that accumulates in E. coli and few other bacteria (4,13,14,16,17,23,26). On the other hand, the EcΔybeY pWSK-SmYbeY strain has markedly reduced 17S rRNA precursor and a significantly increased amount of mature 16S rRNA. ...
... These results suggest that S. meliloti and possibly other alphaproteobacteria are more resilient to heat stress in the absence of YbeY. These results are further supported by similar observations in a recent study on Agrobacterium tumefaciens (26). ...
Single-strand specific endoribonuclease YbeY has been shown to play an important role in the processing of the 3' end of the 16S rRNA in Escherichia coli. Lack of YbeY results in the accumulation of the 17S rRNA precursor. In contrast to a previous report, we show that Sinorhizobium meliloti YbeY exhibits endoribonuclease activity on single-stranded RNA substrate but not on the double-stranded substrate. This study also identifies the previously unknown metal ion involved in YbeY function to be Zn2+ and shows that the activity of YbeY is enhanced when the occupancy of zinc is increased. We have identified a pre-16S rRNA precursor that accumulates in the S. meliloti ΔybeY strain. We also show that ΔybeY mutant of Brucella abortus, a mammalian pathogen, also accumulates a similar pre-16S rRNA. The pre-16S species is longer in alpha-proteobacteria than in gamma-proteobacteria. We demonstrate that the YbeY from E. coli and S. meliloti can reciprocally complement the rRNA processing defect in a ΔybeY mutant of the other organism. These results establish YbeY as a zinc-dependent single-strand specific endoribonuclease that functions in 16S rRNA processing in both alpha- and gamma-proteobacteria.
... In enterohemorrhagic Escherichia coli, YbeY was found to stabilize a type 3 secretion system required for virulence in humans (McAteer et al., 2018). A recent study also found that YbeY was crucial for T-DNA transfer and tumour formation in the plant pathogen Agrobacterium tumefaciens (Möller et al., 2019). ...
... In this study, we reported the characterization of YbeY from Liberibacter asiaticus and established the feasibility of using this protein as a target for the development of antimicrobials. In E. coli, B. abortus, S. meliloti, and A. tumefaciens, YbeY homologues are involved in normal cellular growth as well as antibiotic resistance (β-lactams) and resistance to environmental stress, including temperature, detergents and oxidative stress (Davies and Walker, 2008;Davies et al., 2010;Budnick et al., 2018;Möller et al., 2019). YbeY homologues in Bacillus subtilis and Haemophilus influenzae serve a more critical role and are recognized as essential proteins (Akerley et al., 2002;Kobayashi et al., 2003;Baumgardt et al., 2018). ...
Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeYLas was characterized as an endoribonuclease with activity on 3' and 5' termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeYLas protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site‐directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeYLas. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeYLas. Luteolin was subsequently examined for efficacy against L. asiaticus in HLB‐infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed. This article is protected by copyright. All rights reserved.
Pathogenic bacteria often encounter fluctuating reactive oxygen species (ROS) levels, particularly during host infection, necessitating robust redox-sensing mechanisms for survival. The LysR-type transcriptional regulator (LTTR) OxyR is a widely conserved bacterial thiol-based redox sensor. However, members of the Rhizobiales also encode LsrB, a second LTTR with potential redox-sensing function. This study explores the roles of OxyR and LsrB in the plant-pathogen Agrobacterium tumefaciens. Through single and combined deletions, we observed increased H2O2 sensitivity, underscoring their function in oxidative defense. Genome-wide transcriptome profiling under H2O2 exposure revealed that OxyR and LsrB co-regulate key antioxidant genes, including katG, encoding a bifunctional catalase/peroxidase. Agrobacterium tumefaciens LsrB possesses four cysteine residues potentially involved in redox sensing. To elucidate the structural basis for redox-sensing, we applied single-particle cryo-EM (cryogenic electron microscopy) to experimentally confirm an AlphaFold model of LsrB, identifying two proximal cysteine pairs. In vitro thiol-trapping coupled with mass spectrometry confirmed reversible thiol modifications of all four residues, suggesting a functional role in redox regulation. Collectively, these findings reveal that A. tumefaciens employs two cysteine-based redox sensing transcription factors, OxyR and LsrB, to withstand oxidative stress encountered in host and soil environments.
