Next Article in Journal
Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum
Next Article in Special Issue
Patterns of Genomic Variations in the Plant Pathogen Dickeya solani
Previous Article in Journal
Impact of COVID-19 Infection on Children and Adolescents after Liver Transplantation in a Latin American Reference Center
Previous Article in Special Issue
Unraveling the Uncharacterized Domain of Carocin S2: A Ribonuclease Pectobacterium carotovorum subsp. carotovorum Bacteriocin
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 10
Issue 5
10.3390/microorganisms10051031
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Hfq Is a Critical Modulator of Pathogenicity of Dickeya oryzae in Rice Seeds and Potato Tubers

by
Zurong Shi
1,2,3,†,
Qingwei Wang
1,†,
Shunchang Wang
2,
Chengrun Wang
2,
Lian-Hui Zhang
1,* and
Zhibin Liang
1,*
1
Guangdong Laboratory for Lingnan Modern Agriculture, Guangdong Province Key Laboratory of Microbial Signals and Disease Control, Integrative Microbiology Research Centre, South China Agricultural University, Guangzhou 510642, China
2
School of Biological Engineering, Huainan Normal University, Huainan 232038, China
3
Innovative Institute for Plant Health, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2022, 10(5), 1031; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10051031
Submission received: 21 April 2022 / Revised: 10 May 2022 / Accepted: 13 May 2022 / Published: 16 May 2022
(This article belongs to the Special Issue Dickeya and Pectobacterium: Ecology, Pathology and Plant Protection 2.0)
Browse Figures
Versions Notes

Abstract

:
The frequent outbreaks of soft-rot diseases caused by Dickeya oryzae have emerged as severe problems in plant production in recent years and urgently require the elucidation of the virulence mechanisms of D. oryzae. Here, we report that Hfq, a conserved RNA chaperone protein in bacteria, is involved in modulating a series of virulence-related traits and bacterial virulence in D. oryzae EC1. The findings show that the null mutation of the hfqEC1 gene totally abolished the production of zeamine phytotoxins and protease, significantly attenuated the production of two other types of cell wall degrading enzymes, i.e., pectate lyase and cellulase, as well as attenuating swarming motility, biofilm formation, the development of hypersensitive response to Nicotiana benthamiana, and bacterial infections in rice seeds and potato tubers. QRT-PCR analysis and promoter reporter assay further indicated that HfqEC1 regulates zeamine production via modulating the expression of the key zeamine biosynthesis (zms) cluster genes. Taken together, these findings highlight that the Hfq of D. oryzae is one of the key regulators in modulating the production of virulence determinants and bacterial virulence in rice seeds and potato tubers.

1. Introduction

Dickeya spp. are destructive plant pathogenic bacteria worldwide, which can cause soft-rot diseases in both monocotyledonous and dicotyledonous plants [1]. Rice foot rot disease, caused by one of the members of Dickeya spp., i.e., D. oryzae, has posed a great threat to not only rice production in China and some other Asian countries, including India, Indonesia, and Japan [2,3,4], but also to potato production in Australia [5]. Recent works unveiled the virulence determinants of D. oryzae, including phytotoxic zeamines, motility, cell wall–degrading enzymes (CWDEs), and biofilm formation [6,7,8]. Production of these determinants is regulated at different bacterial growth stages by the AHL quorum-sensing signal [2], putrescine signal [8], transcriptional regulators [9,10], the two-component signal transduction system [11,12], and the second messenger cyclic-di-GMP [13,14]. Until now, the contributions of small RNAs (sRNAs) and their chaperone proteins to the pathogenicity of D. oryzae are still unclear.
Post-transcriptional regulation in bacteria is typically mediated by the base-pairing interactions between regulatory small RNAs (sRNAs) and their target mRNA transcripts, the outcome of which changes either translational efficiency, or mRNA stability, or both [15,16]. The interactions between sRNAs and their targets are often assisted by the RNA chaperone protein, i.e., Hfq. Hfq was firstly characterized in Escherichia coli as an essential host factor required for the replication of RNA bacteriophage Qbeta [17]. Subsequent studies unveiled that Hfq is a highly conserved, global sRNA chaperone in Gram-positive and -negative bacterial species [18,19], broadly involved in the regulation of bacterial physiology and metabolism, including membrane protein composition, stress tolerance, motility, and biofilm formation [20,21].
Previous works have been carried out to examine the role of Hfq in the growth of pathogenic bacteria. Mutation of hfq can impair the in vitro multiplication of bacterial pathogens belonging to E. coli [22], Vibrio cholerae [23], Yersinia pseudotuberculosis [24], and Dickeya dadantii [25] but has no effect on the growth of Xanthomonas campestris pv. campestris [26]. In addition, recent advances indicate that Hfq contributes to the pathogenicity of plant pathogens belonging to Agrobacterium tumefaciens [27], Pantoea ananatis [28], Pectobacterium carotovorum [29], X. campestris pv. campestris [26], and Erwinia amylovora [30]. However, Hfq is not implicated in the virulence of Xanthomonas campestris pv. vesicatoria and Xanthomonas oryzae pv. oryzae [31,32]. In D. dadantii 3937, Hfq was found to be essential for regulation of bacterial virulence. Inactivation of hfq strongly reduced bacterial motility and production of CWDEs, i.e., cellulose (Cel), pectate lyase (Pel), and protease (Prt), and compromised the capacity of D. dadantii 3937 to cause soft rot in chicory leaves [25].
Considering the great contributions of Hfq to bacterial growth, the production of virulence factors, and virulence in D. dandatii, we hypothesize that Hfq also plays significant roles in the virulence of D. oryzae. To elucidate the roles of Hfq in D. oryzae, in this study, we constructed an in-frame deletion mutant of hfq in D. oryzae EC1. Then, we assessed the influence of the inactivation of hfq on bacterial growth, the production of virulence-related traits, and bacterial virulence in rice seeds and potato tuber slices in D. oryzae EC1, steps which aimed to determine whether Hfq is an important modulator in the virulence of D. oryzae and to expand understanding of the roles of Hfq in different Dickeya species.

2. Materials and Methods

2.1. Bacterial Strains, Plasmids, and Cultural Conditions

The bacterial strains and plasmids used in this study are listed in Supplementary Table S1. D. oryzae EC1 and its derivatives were cultivated at 28 °C in minimal medium (MM), LS5 medium, and Luria–Bertani (LB) medium, as indicated [9]. The E. coli strains were grown at 37 °C in LB medium. Antibiotics were added to the medium at the following final concentrations when required: ampicillin, 100 µg/mL; streptomycin, 50 µg/mL; kanamycin, 50 µg/mL.

2.2. Construction of the HfqEC1 Deletion Mutant and Complementation Strain

The method used for constructing the deletion mutant of hfq gene in D. oryzae EC1 (hfqEC1) and the corresponding complementation strain were previously described [8]. Briefly, fragments containing about a 500 bp upstream and downstream region of the hfqEC1 were amplified from the genomic DNA of wild-type strain EC1, respectively, and fused by overlapping extension PCR with the forward primer of the upstream fragment (Primer A-1, Supplementary Table S2) and the reverse primer of the downstream fragment (Primer A-4, Supplementary Table S2). The PCR product was purified, digested with restriction enzymes, and ligated to the vector pKNG101 digested with the same enzymes. The resultant construct was transformed into E. coli CC118 competent cells by heat shock at 42 °C and introduced into wild-type strain EC1 through triparental mating with the helper strain HB101 (pRK2013). The desired mutants were screened on MM agar plates containing 5% (wt/vol) sucrose and confirmed by PCR and DNA sequencing. For complementation, the coding sequence of hfqEC1 was amplified from the genomic DNA of wild-type strain EC1 with the primer pairs HB-A-F and HB-A-R (Supplementary Table S2). The PCR product was digested with restriction enzymes and introduced into pBBR1-MCS4 digested with the same enzymes. The desired complementation construct was introduced into the ΔhfqEC1 by triparental mating and confirmed by PCR. The gene is expressed under the control of the lac promoter in pBBR1-MCS4.

2.3. Cell Wall–Degrading Enzyme Activities

The methods used for measuring the activity of Cel, Pel, and Prt were previously described, with minor modifications [13]. The compositions of media used for enzyme activity assays were as follows: Cel medium—carboxymethyl cellulose 1.0 g/L, Na3PO4 3.8 g/L, agarose 8.0 g/L, pH 7.0; Pel medium—polygalacturonic acid 10 g/L, yeast extract 10 g/L, CaCl2 0.1125 g/L, Tris-HCl 100 mM, agarose 8 g/L, pH 8.5; and Prt medium—the LB medium containing equal volume of 1% (wt/vol) skimmed milk. For semi-quantitative assays, wells of 5 mm were punched in plates. Twenty microliters of bacterial cell cultures (OD600 of 1.5) were added to the wells in plates with incubation at 28 °C. In Pel assay, the plates were treated with 1 N HCl after the incubation for 11 h. In Cel assay, the plates were developed by 0.1% (wt/vol) Congo Red staining and sequentially decolorized by 1 M NaCl. Halos around the wells became visible in Prt assay plates after 20 h without any further treatment.

2.4. Motility Assay and Biofilm Formation

Motility assay was performed by using a method described previously [8]. Briefly, swimming motility was elucidated in the semi-solid medium plate containing about 15 mL of semi-solid Bacto tryptone agar medium (Bacto tryptone 10 g/L, NaCl 5 g/L, and agar 3 g/L). An aliquot of 2 μL overnight bacterial culture was inoculated into 15 mL of semi-solid agar medium in the plates. The diameters of swimming motility were measured after the incubation at 28 °C for 24 h. In swarming motility assay, bacterial cells were inoculated in the middle of the plate, containing 15 mL of semi-solid agar medium (Tryptone 10 g/L, NaCl 5 g/L, and agar 4 g/L). The plates were incubated at 28 °C for 18 h, and the diameters of the swarming motility were measured.
To quantify the biofilm formation, overnight bacterial culture was 1:100 diluted into the super optimal broth plus glycerol (SOBG) medium (Tryptone 20 g/L, yeast extract 5 g/L, MgSO4 2.4 g/L, NaCl 0.5 g/L, KCl 0.186 g/L, and glycerol 2 g/L) in the 96-well plates. The plates were incubated at 28 °C with shaking at 150 rpm for 18 h. After the incubation, bacterial cell cultures were removed and an aliquot of 200 μL crystal violet (0.1% wt/vol) was added into each well for a 15 min staining. After the staining, the unbound crystal violet was removed and the wells were rinsed three times with water. The remaining crystal violet in each well was decolorized with 200 μL of 95% ethanol after dryness and quantified by measuring the absorbance at 570 nm.

2.5. Zeamine Production Assay

Zeamines are a family of structurally related phytotoxins required for the virulence of D. oryzae EC1 in rice seeds and potato tubers [6,7,33]. In addition, they are also potent antibiotics having broad activity against various organisms, including bacteria, fungi, and nematodes [33,34,35]. Zeamine production in the wild-type strain EC1 and the hfqEC1 deletion mutant were determined by measuring the antibiotic activity of zeamines against E. coli DH5α [7]. Briefly, bacterial cell cultures grown overnight in LS5 medium to an OD600 of 1.5 were filtered. An aliquot of 30 μL cell-free supernatants was added into the wells in bioassay plates, in which the 15 mL LB agar medium was overlaid with 5 mL of 1% agarose containing about 1.0 × 108 cells of E. coli DH5α. The plates were incubated at 37 °C for 18 h to measure the radius of the visible clear zone surrounding the wells in order to determine the production of zeamines in D. oryzae EC1 and hfqEC1 mutant.

2.6. RNA Extraction and Quantitative Real-Time Reverse-Transcription PCR (qRT-PCR) Analysis

The wild-type strain EC1 and hfqEC1 mutant were grown in LS5 medium to an OD600 about 1.5. The RNA samples were prepared using the SV total RNA isolated system kit (Promega, Beijing, China) and further purified using the RNA clean kit (Qiagen, Hilden, Germany). The cDNA synthesis was performed by using StarScript II first-strand cDNA synthesis Mixing followed the manufacturer’s instructions (GenStar Biosolutions, Beijing, China). The qRT-PCR analysis was conducted on a Quantstudio 6 Flex system using PowerUp SYBR green master mix (Thermo Fisher Scientific, Waltham, MA, USA) with the primers listed in Supplementary Table S2 and followed cycle profile: 1 cycle at 50 °C for 2 min and 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Data were analyzed using the 2−ΔΔCT method, as previously described [36].

2.7. Pathogenicity Assay on Potato Tuber Slices

Pathogenicity assay on potato tubers was performed by using methods described previously [7]. Briefly, potato tubers were sliced evenly to 5 mm in thickness after washing and surface disinfestation. An aliquot of 2 μL of bacterial cell cultures at an OD600 of 1.5 was added to the center of each sliced potato tuber. The potato tuber slices were then incubated at 28 °C and observed regularly for symptom development.

2.8. Rice Seed Germination Assay

The rice seed germination assay was conducted as previously described [2]. Briefly, overnight bacterial cultures were diluted in 10-fold series and the CFU (colony-forming unit) of each dilution was determined by using the plate counting assay. Thirty seeds of rice variety CO39 were added to 9 mL of bacterial dilution and incubated at room temperature (25 °C) for 5 h. After the incubation, the rice seeds were washed three times with sterilized water and subsequently transferred onto the moistened filter papers in petri dishes for incubation at 28 °C with a 16 h light and 8 h dark cycle. Rice seeds incubated with the same amount of sterilized water were considered as the control. The sterilized water was added to the filter papers during incubation when necessary. After incubation for 7 d, the rate of seed germination was determined.

2.9. Hypersensitive Response Assay

The hypersensitive responses (HR) of D. oryzae EC1 and its derivatives were tested on the nonhost plant Nicotiana benthamiana. The upper surface of leaf was inoculated by infiltrating approximately 5 μL of bacterial cell cultures (OD600 = 0.05, 2.0 × 104 CFU/mL) using a 1 mL blunt-end plastic syringe. The inoculated plants were incubated in a greenhouse with a 12 h day-and-night cycle at 28 °C. HR symptoms were photographed 24 h post-inoculation. At least three plants were inoculated in each experiment.

2.10. Statistical Analysis

Experiments were individually performed at least three times with three replicates each time. Statistical comparison was performed by using Student’s t test in GraphPad Prism 5.0 software (GraphPad, La Jolla, CA, USA). A p value of less than 0.05 was considered significant.

3. Results

3.1. Identification of Hfq in D. oryzae EC1

Homology blast and sequence alignment unveiled that a putative hfq gene, named hfqEC1, exhibits 82% sequence similarity at the amino acid level (NCBI accession no. WP_012886271) compared with the hfq in E. coli (NCBI accession no. ACE63256.1) and is present in D. oryzae EC1 (Figure 1A). To determine the role of hfqEC1 in the growth of D. oryzae EC1, we compared the growth patterns of wild-type strain EC1 and ΔhfqEC1 in rich medium (LB) and the minimal medium (MM), respectively. The results showed that the hfqEC1 mutant had a comparable growth pattern to the wild-type strain EC1 in both rich and minimal media (Figure 1B,C), which suggests hfqEC1 is not required for the growth of D. oryzae EC1 in vitro.

3.2. Deletion of HfqEC1 Decreases the Production of Extracellular Degrading Enzymes in D. oryzae EC1

To elucidate whether hfqEC1 is responsible for the production of CWDEs in D. oryzae EC1, the production of CWDEs in wild-type strain EC1 and hfqEC1 mutant were compared with the agar plates with substrates of Pel, Cel, and Prt. The results showed that the production of Cel and Pel in the hfqEC1 mutant decreased by 17% and 21%, respectively, compared with those in wild-type strain EC1 (Figure 2A,B). Notably, inactivation of hfqEC1 totally abolished the production of Prt in D. oryzae EC1 (Figure 2C). The complementation analysis showed that in trans expression of hfqEC1 could restore the production of all these three types of CWDEs (Figure 2A–C). These results indicate that the HfqEC1 is required for the production of CWDEs in D. oryzae EC1, especially Prt.

3.3. HfqEC1 Contributes to Swarming Motility and Biofilm Formation in D. oryzae EC1

To elucidate whether HfqEC1 play key roles in motility and biofilm formation in D. oryzae EC1, the motility and biofilm-forming capacity of wild-type strain EC1 and the hfqEC1 deletion mutant were compared. The result showed that the ΔhfqEC1 displayed a comparable swimming motility but a decreased swarming motility compared with the wild-type strain EC1 (Figure 3A,B). Consistently, the crystal violet staining assay performed in the 96-well plates revealed that the biofilm formation in the hfqEC1 mutant represented a significant decrease compared with that in wild-type strain EC1 (Figure 3C). All these results indicate that HfqEC1 contributes to the regulation of swarming motility and biofilm formation in D. oryzae EC1.

3.4. HfqEC1 Regulates Zeamine Production through Modulating the Expression of Key Zms Cluster Genes

To elucidate whether HfqEC1 is required for the production of zeamines in D. oryzae EC1, zeamine assays were performed on wild-type strain EC1 and the hfqEC1 mutant. Intriguingly, the result showed that inactivation of hfqEC1 resulted in a complete loss of zeamine production in D. oryzae EC1. Conversely, in trans expression of hfqEC1 could restore the zeamine production of the hfqEC1 mutant (Figure 4A). This finding shows that HfqEC1 plays a key role in modulating zeamine production in D. oryzae EC1. To elucidate whether the zeamine production conferred by HfqEC1 relies on the transcriptional regulation of key zms cluster genes [37], the expression of zmsA–G and zmsI–K was determined by qRT-PCR assay when bacterial strains were cultured in the medium optimized for zeamine production (LS5 medium). The result indicated that expression of the key zms cluster genes in hfqEC1 mutant, especially the zmsA, was decreased to different degrees compared with that in the wild-type strain EC1 at an OD600 of 1.5, where zeamines are largely produced by D. oryzae EC1 [38] (Figure 4B). In addition, the GFP promoter reporter fusion assay further showed that expression of zmsD in wild-type strain EC1 was continuously increased and higher than that in the hfqEC1 mutant at an OD600 from 0.8 to 1.6 in LS5 medium (Figure 4C). These results indicate that HfqEC1 controls zeamine production mainly through regulating the expression of key zms cluster genes.

3.5. HfqEC1 Is Required for the Virulence of D. oryzae EC1

To determine whether HfqEC1 is involved in the regulation of the pathogenicity of D. oryzae EC1, we compared the infections of wild-type strain EC1 and the hfqEC1 mutant in rice seeds and potato tuber slices. The results showed that potato tuber slices inoculated with the hfqEC1 mutant had substantially reduced rotting areas compared with those inoculated with the wild-type strain EC1 at 48 h post-inoculation (Figure 5A). Similarly, the wild-type strain EC1 was much more virulent than the hfqEC1 mutant on rice seed germination, showing about an 82% inhibition rate when rice seeds were treated with 10 bacterial cells per mL and total inhibition at 100 bacterial cells per mL. Compared with the wild-type strain EC1, the hfqEC1 mutant was unable to inhibit rice seed germination at a concentration range from 10 to 10,000 bacterial cells per mL, only showing about a 73% inhibition rate when the bacterial inoculation was increased to 108 cells per mL (Figure 5B). Conversely, in trans expression of hfqEC1 in hfqEC1 mutant restored bacterial infective capacity in rice seed germination and potato tuber slices (Figure 5A,B). Taken together, these findings indicate that HfqEC1 is required for the virulence of D. oryzae EC1 in both monocotyledonous and dicotyledonous plants.
To determine whether HfqEC1 is required for triggering hypersensitive response (HR) on nonhost plants in D. oryzae EC1, the HR symptoms on N. benthamiana leaves developed by wild-type strain EC1 and the hfqEC1 mutant were compared. The results showed that HR lesion developed by the hfqEC1 mutant was defective in size compared with that developed by wild-type strain EC1 after 24 h inoculation (Figure 5C), suggesting that HfqEC1 is essential for D. oryzae EC1 to trigger HR in nonhost plant N. benthamiana.

4. Discussion

The Hfq proteins are conserved and essential regulators for regulating the production of divergent virulence factors in a large number of bacterial pathogens. In this study, we systematically elucidated the roles of HfqEC1 in the production of virulence factors and bacterial virulence in D. oryzae EC1. Inactivation of the hfqEC1 totally abolished the production of Prt and zeamines in D. oryzae EC1 (Figure 2C and Figure 4A) and dramatically reduced the pathogenicity of D. oryzae EC1 in potato tubers and rice seeds (Figure 5A,B). QRT-PCR and promoter reporter assay further showed that HfqEC1 modulates the production of zeamines through transcriptional regulation of key zms cluster genes. The transcriptional regulation of zms cluster genes by HfqEC1 may suggest cooperation between HfqEC1 and other transcriptional regulatory mechanisms through sRNA-based post-transcription regulation in D. oryzae EC1.
Previous studies indicated that inactivation of hfq could cause reduced bacterial growth rates in a large proportion of bacterial species, including D. dadantii, P. carotovorum, A. tumefaciens, P. ananatis, and E. amylovora [25,27,28,29,30]. However, despite the relatively close taxonomic relationship between D. dadantii and D. oryzae, similar to bacterial strain belong to X. campestris pv. campestris [26], we found that the growth rates of hfqEC1 mutant were comparable to those of the wild-type strain EC1 in the selected culture conditions (Figure 1B,C), which suggests the defective phenotype of the hfqEC1 mutant is not related to the proposed involvement of HfqEC1 in the growth of D. oryzae EC1, and Hfq plays differential roles in different Dickeya species.
Zeamines are crucial to the infection of D. oryzae EC1 in rice seeds [6,7]. In this study, we found the capacity of D. oryzae EC1 for inhibiting rice seed germination was largely impaired at each inoculation concentration after inactivation of hfqEC1 (Figure 5B). This resulted from the contribution of HfqEC1 to the production of zeamines (Figure 4A). The role of Hfq in regulation of zeamine production was also reported in a biocontrol bacterial isolate, i.e., Serratia plymuthica A153 [34]. Inactivation of hfq in S. plymuthica A153 totally abolished the zeamine production. All these factors suggest the conserved regulatory role of Hfq in zeamine production across different bacterial species. Hfq is closely associated with the RsmA/RsmB signaling pathway in the regulation of biofilm formation in E. coli [39] and the production of CWDEs in D. dadantii 3937 [40]. In D. oryzae EC1, our recent work unveiled that the regulons of rmsB, i.e., TzpS-TzpA, which are homologous to GacS-GacA, are also implicated in the production of zeamines through transcriptional regulation of zms cluster genes [12]. In this study, we found that HfqEC1 regulated the expression of zms cluster genes at transcriptional level. The proposed cross-talk among TzpS-TzpA, RsmA/RsmB, and HfqEC1 in the transcriptional regulation of zms gene expression is intriguing and requires further elucidation.
The CWDEs are essential for plant pathogens to develop soft-rot symptoms in plants. The regulatory roles of Hfq proteins on the production of CWDEs were documented in soft-rot plant pathogens belonging to Dickeya and Pectobacterium [25,29]. In this work, we showed that production of Prt was totally abolished in the hfqEC1 deletion mutant (Figure 2C). Moreover, significant reductions on the production of Pel and Cel were also noticed in the hfqEC1 mutant compared with wild-type strain EC1 (Figure 2A,B). Consistent with these findings, the null mutation of hfqEC1 attenuated the rotting areas on potato tubers (Figure 5A). Compared with the hfq in well-studied D. dadantii 3937, which plays a major role in the production of Pel [25], we found that hfqEC1 was largely involved in the production of Prt but not Pel (Figure 2B,C), which suggests Hfq proteins confer divergent regulatory networks for the production of CWDEs in D. dadantii and D. oryzae.
The HR is a phenotype of programmed cell death that bacterial pathogens can induce through the type-III secretion system (T3SS) in nonhost plants [41,42]. Although the Hfq proteins in plant pathogens are frequently associated with the production of CWDEs, motility, and biofilm formation, they did not commonly contribute to the induction of HR in plant pathogens. The association between Hfq and HR developed by plant pathogens are so far reported only in a few bacterial species, including E. amylovora and D. dadantii. In E. amylovora, Hfq regulates the translocation and secretion of the effector DspE [30]. In D. dadantii 3937, Hfq modulates the expression of T3SS though positively regulating the expression of rsmB at the post-transcriptional level [40]. In this study, we found that the null mutation of hfqEC1 not only dramatically attenuated the virulence of D. oryzae EC1 in host plants, i.e., potato and rice, but also abolished the development of HR symptoms in nonhost plant N. benthamiana (Figure 4), which suggests a potential link between Hfq and expression of the type-III secretion system in D. oryzae EC1.

5. Conclusions

In summary, this study unveiled the key role of the RNA chaperone protein HfqEC1 in the production of virulence-related traits, particularly Prt and zeamine phytotoxins, both of which are key virulence factors required for virulence of D. oryzae EC1 in rice seeds and potato tubers. In addition, this study highlights the divergence of regulatory networks mediated by Hfq proteins for bacterial growth and production of CWDEs in different Dickeya species. In a further study, it would be intriguing to elucidate the HfqEC1-dependent sRNA regulatory network and its cross-link with the previously determined transcriptional regulatory mechanisms required for zeamine production in D. oryzae EC1.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/microorganisms10051031/s1, Table S1. Strains and plasmids used in this study. Table S2. Primers used in this study.

Author Contributions

Conceptualization, Z.S., L.-H.Z. and Z.L.; methodology, Q.W.; validation, S.W. and C.W.; formal analysis, Z.S. and Q.W.; investigation, Z.S. and Q.W.; resources, L.-H.Z. and Z.L.; writing—original draft preparation, Z.S. and Z.L.; writing—review and editing, Q.W. and L.-H.Z.; supervision, L.-H.Z.; funding acquisition, Z.S., L.-H.Z. and Z.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Anhui Province (2108085QC118), the Guangzhou Science and Technology Plan (201804020066; 202102080488), the Guangdong Forestry Science and Technology Innovation Project (2018KJCX009; 2020KJCX009), the Key Realm R&D Program of Guangdong Province (2018B020205003; 2020B0202090001), the Guangdong Basic and Applied Basic Research Foundation (2020A1515110465), and the National Natural Science Foundation of China (32000085).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genomic sequence of D. oryzae EC1 in NCBI is accessible with No. NZ_CP006929.1.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Reverchon, S.; Nasser, W. Dickeya Ecology, Environment Sensing and Regulation of Virulence Programme. Environ. Microbiol. Rep. 2013, 5, 622–636. [Google Scholar] [CrossRef] [PubMed]
  2. Hussain, M.B.; Zhang, H.B.; Xu, J.L.; Liu, Q.; Jiang, Z.; Zhang, L.H. The Acyl-Homoserine Lactone-Type Quorum-Sensing System Modulates Cell Motility and Virulence of Erwinia chrysanthemi pv. zeae. J. Bacteriol. 2008, 190, 1045–1053. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Goto, M. Bacterial Foot Rot of Rice Caused by a Strain of Erwinia chrysanthemi. Phytopathology 1979, 69, 213. [Google Scholar] [CrossRef] [Green Version]
  4. Liu, Q.G.; Zhang, Q.; Wei, C.D. Advances in Research of Rice Bacterial Foot Rot. Sci. Agric. Sin. 2013, 46, 2923–2931. [Google Scholar]
  5. Pritchard, L.; Humphris, S.; Saddler, G.S.; Elphinstone, J.G.; Pirhonen, M.; Toth, I.K. Draft Genome Sequences of 17 Isolates of the Plant Pathogenic Bacterium Dickeya. Genome Announc. 2013, 1, e00978-13. [Google Scholar] [CrossRef] [Green Version]
  6. Cheng, Y.; Liu, X.; An, S.; Chang, C.; Zou, Y.; Huang, L.; Zhong, J.; Liu, Q.; Jiang, Z.; Zhou, J.; et al. A Nonribosomal Peptide Synthase Containing a Stand-Alone Condensation Domain Is Essential for Phytotoxin Zeamine Biosynthesis. Mol. Plant-Microbe Interact. 2013, 26, 1294–1301. [Google Scholar] [CrossRef] [Green Version]
  7. Zhou, J.; Zhang, H.; Wu, J.; Liu, Q.; Xi, P.; Lee, J.; Liao, J.; Jiang, Z.; Zhang, L.H. A Novel Multidomain Polyketide Synthase Is Essential for Zeamine Production and the Virulence of Dickeya zeae. Mol. Plant-Microbe Interact. 2011, 24, 1156–1164. [Google Scholar] [CrossRef] [Green Version]
  8. Shi, Z.; Wang, Q.; Li, Y.; Liang, Z.; Xu, L.; Zhou, J.; Cui, Z.; Zhang, L.H. Putrescine Is an Intraspecies and Interkingdom Cell-Cell Communication Signal Modulating the Virulence of Dickeya zeae. Front. Microbiol. 2019, 10, 1950. [Google Scholar] [CrossRef] [Green Version]
  9. Zhou, J.N.; Zhang, H.B.; Lv, M.F.; Chen, Y.F.; Liao, L.S.; Cheng, Y.Y.; Liu, S.Y.; Chen, S.H.; He, F.; Cui, Z.N.; et al. SlyA Regulates Phytotoxin Production and Virulence in Dickeya zeae EC1. Mol. Plant Pathol. 2016, 17, 1398–1408. [Google Scholar] [CrossRef]
  10. Lv, M.; Chen, Y.; Liao, L.; Liang, Z.; Shi, Z.; Tang, Y.; Ye, S.; Zhou, J.; Zhang, L. Fis Is a Global Regulator Critical for Modulation of Virulence Factor Production and Pathogenicity of Dickeya zeae. Sci. Rep. 2018, 8, 341. [Google Scholar] [CrossRef] [Green Version]
  11. Lv, M.; Hu, M.; Li, P.; Jiang, Z.; Zhang, L.H.; Zhou, J. A Two-Component Regulatory System VfmIH Modulates Multiple Virulence Traits in Dickeya zeae. Mol. Microbiol. 2019, 111, 1493–1509. [Google Scholar] [CrossRef] [PubMed]
  12. Chen, Y.; Li, Y.; Zhu, M.; Lv, M.; Liu, Z.; Chen, Z.; Huang, Y.; Gu, W.; Liang, Z.; Chang, C.; et al. The GacA-GacS Type Two-Component System Modulates the Pathogenicity of Dickeya oryzae EC1 Mainly by Regulating the Production of Zeamines. Mol. Plant. Microbe. Interact. 2022, 35, 369–379. [Google Scholar] [CrossRef]
  13. Chen, Y.; Lv, M.; Liao, L.; Gu, Y.; Liang, Z.; Shi, Z.; Liu, S.; Zhou, J.; Zhang, L. Genetic Modulation of c-di-GMP Turnover Affects Multiple Virulence Traits and Bacterial Virulence in Rice Pathogen Dickeya zeae. PLoS ONE 2016, 11, e0165979. [Google Scholar] [CrossRef] [Green Version]
  14. Chen, Y.; Zhou, J.; Lv, M.; Liang, Z.; Parsek, M.R.; Zhang, L.H. Systematic Analysis of c-di-GMP Signaling Mechanisms and Biological Functions in Dickeya zeae EC1. MBio 2020, 11, e02993-20. [Google Scholar] [CrossRef] [PubMed]
  15. Wagner, E.G.H.; Romby, P. Small RNAs in Bacteria and Archaea: Who They Are, What They Do, and How They Do It. Adv. Genet. 2015, 90, 133–208. [Google Scholar] [CrossRef]
  16. Waters, L.S.; Storz, G. Regulatory RNAs in Bacteria. Cell 2009, 136, 615–628. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Franze de Fernandez, M.T.; Eoyang, L.; August, J.T. Factor Fraction Required for the Synthesis of Bacteriophage Qbeta-RNA. Nature 1968, 219, 588–590. [Google Scholar] [CrossRef] [PubMed]
  18. Updegrove, T.B.; Zhang, A.; Storz, G. 2016 Hfq: The Flexible RNA Hitchmaker. Physiol. Behav. 2016, 176, 100–106. [Google Scholar] [CrossRef]
  19. Feliciano, J.R.; Grilo, A.M.; Guerreiro, S.I.; Sousa, S.A.; Leitão, J.H. Hfq: A Multifaceted RNA Chaperone Involved in Virulence. Future Microbiol. 2016, 11, 137–151. [Google Scholar] [CrossRef]
  20. Chao, Y.; Vogel, J. The Role of Hfq in Bacterial Pathogens. Curr. Opin. Microbiol. 2010, 13, 24–33. [Google Scholar] [CrossRef]
  21. Mizrahi, S.P.; Elbaz, N.; Argaman, L.; Altuvia, Y.; Katsowich, N.; Socol, Y.; Bar, A.; Rosenshine, I.; Margalit, H. The Impact of Hfq-Mediated sRNA-mRNA Interactome on the Virulence of Enteropathogenic Escherichia coli. Sci. Adv. 2021, 7, eabi8228. [Google Scholar] [CrossRef] [PubMed]
  22. Tsui, H.C.; Leung, H.C.; Winkler, M.E. Characterization of Broadly Pleiotropic Phenotypes Caused by an Hfq Insertion Mutation in Escherichia coli K-12. Mol. Microbiol. 1994, 13, 35–49. [Google Scholar] [CrossRef] [PubMed]
  23. Ding, Y.; Davis, B.M.; Waldor, M.K. Hfq Is Essential for Vibrio cholerae Virulence and Downregulates σE Expression. Mol. Microbiol. 2004, 53, 345–354. [Google Scholar] [CrossRef] [PubMed]
  24. Schiano, C.A.; Bellows, L.E.; Lathem, W.W. The Small RNA Chaperone Hfq Is Required for the Virulence of Yersinia pseudotuberculosis. Infect. Immun. 2010, 78, 2034–2044. [Google Scholar] [CrossRef] [Green Version]
  25. Leonard, S.; Villard, C.; Nasser, W.; Reverchon, S.; Hommais, F. RNA Chaperones Hfq and ProQ Play a Key Role in the Virulence of the Plant Pathogenic Bacterium Dickeya dadantii. Front. Microbiol. 2021, 12, 687484. [Google Scholar] [CrossRef]
  26. Lai, J.L.; Tang, D.J.; Liang, Y.W.; Zhang, R.; Chen, Q.; Qin, Z.P.; Ming, Z.H.; Tang, J.L. The RNA Chaperone Hfq Is Important for the Virulence, Motility and Stress Tolerance in the Phytopathogen Xanthomonas campestris. Environ. Microbiol. Rep. 2018, 10, 542–554. [Google Scholar] [CrossRef] [Green Version]
  27. Wilms, I.; Möller, P.; Stock, A.M.; Gurski, R.; Lai, E.M.; Narberhaus, F. Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens. J. Bacteriol. 2012, 194, 5209–5217. [Google Scholar] [CrossRef] [Green Version]
  28. Shin, G.Y.; Schachterle, J.K.; Shyntum, D.Y.; Moleleki, L.N.; Coutinho, T.A.; Sundin, G.W. Functional Characterization of a Global Virulence Regulator Hfq and Identification of Hfq-Dependent sRNAs in the Plant Pathogen Pantoea ananatis. Front. Microbiol. 2019, 10, 2075. [Google Scholar] [CrossRef]
  29. Wang, C.; Pu, T.; Lou, W.; Wang, Y.; Gao, Z.; Hu, B.; Fan, J. Hfq, a RNA Chaperone, Contributes to Virulence by Regulating Plant Cell Wall-Degrading Enzyme Production, Type VI Secretion System Expression, Bacterial Competition, and Suppressing Host Defense Response in Pectobacterium carotovorum. Mol. Plant-Microbe Interact. 2018, 31, 1166–1178. [Google Scholar] [CrossRef] [Green Version]
  30. Zeng, Q.; McNally, R.R.; Sundin, G.W. Global Small RNA Chaperone Hfq and Regulatory Small RNAs Are Important Virulence Regulators in Erwinia amylovora. J. Bacteriol. 2013, 195, 1706–1717. [Google Scholar] [CrossRef] [Green Version]
  31. Liang, H.; Zhao, Y.T.; Zhang, J.Q.; Wang, X.J.; Fang, R.X.; Jia, Y.T. Identification and Functional Characterization of Small Non-Coding RNAs in Xanthomonas oryzae pathovar oryzae. BMC Genom. 2011, 12, 87. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Schmidtke, C.; Abendroth, U.; Brock, J.; Serrania, J.; Becker, A.; Bonas, U. Small RNA SX13: A Multifaceted Regulator of Virulence in the Plant Pathogen Xanthomonas. PLoS Pathog. 2013, 9, e1003626. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Wu, J.; Zhang, H.B.; Xu, J.L.; Cox, R.J.; Simpson, T.J.; Zhang, L.H. 13C Labeling Reveals Multiple Amination Reactions in the Biosynthesis of a Novel Polyketide Polyamine Antibiotic Zeamine from Dickeya zeae. Chem. Commun. 2010, 46, 333–335. [Google Scholar] [CrossRef] [PubMed]
  34. Hellberg, J.E.E.U.; Matilla, M.A.; Salmond, G.P.C. The Broad-Spectrum Antibiotic, Zeamine, Kills the Nematode Worm Caenorhabditis elegans. Front. Microbiol. 2015, 6, 137. [Google Scholar] [CrossRef] [Green Version]
  35. Liao, L.; Zhou, J.; Wang, H.; He, F.; Liu, S.; Jiang, Z.; Chen, S.; Zhang, L.H. Control of Litchi Downy Blight by Zeamines Produced by Dickeya zeae. Sci. Rep. 2015, 5, 15719. [Google Scholar] [CrossRef] [Green Version]
  36. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  37. Zhou, J.; Cheng, Y.; Lv, M.; Liao, L.; Chen, Y.; Gu, Y.; Liu, S.; Jiang, Z.; Xiong, Y.; Zhang, L. The Complete Genome Sequence of Dickeya zeae EC1 Reveals Substantial Divergence from Other Dickeya Strains and Species. BMC Genom. 2015, 16, 571. [Google Scholar] [CrossRef] [Green Version]
  38. Liang, Z.; Huang, L.; He, F.; Zhou, X.; Shi, Z.; Zhou, J.; Chen, Y.; Lv, M.; Chen, Y.; Zhang, L.-H. A Substrate-Activated Efflux Pump, DesABC, Confers Zeamine Resistance to Dickeya zeae. MBio 2019, 10, e00713-19. [Google Scholar] [CrossRef] [Green Version]
  39. Jørgensen, M.G.; Thomason, M.K.; Havelund, J.; Valentin-Hansen, P.; Storz, G. Dual Function of the McaS Small RNA in Controlling Biofilm Formation. Genes Dev. 2013, 27, 1132–1145. [Google Scholar] [CrossRef] [Green Version]
  40. Yuan, X.; Zeng, Q.; Khokhani, D.; Tian, F.; Severin, G.B.; Waters, C.M.; Xu, J.; Zhou, X.; Sundin, G.W.; Ibekwe, A.M.; et al. A Feed-Forward Signalling Circuit Controls Bacterial Virulence through Linking cyclic di-GMP and Two Mechanistically Distinct sRNAs, ArcZ and RsmB. Environ. Microbiol. 2019, 21, 2755–2771. [Google Scholar] [CrossRef]
  41. Balint-Kurti, P. The Plant Hypersensitive Response: Concepts, Control and Consequences. Mol. Plant Pathol. 2019, 20, 1163–1178. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Tang, X.; Xiao, Y.; Zhou, J.M. Regulation of the Type III Secretion System in Phytopathogenic Bacteria. Mol. Plant-Microbe Interact. 2006, 19, 1159–1166. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Microorganisms 10 01031 g001 550
Figure 1. Gene organization of hfqEC1 and its influence on bacterial growth in Dickeya oryzae. (A) Gene organization of hfqEC1. Growth patterns of the wild-type strain EC1 and the hfqEC1 deletion mutant were measured in Luria–Bertani (LB) medium (B) and minimal medium (MM) (C).
Figure 1. Gene organization of hfqEC1 and its influence on bacterial growth in Dickeya oryzae. (A) Gene organization of hfqEC1. Growth patterns of the wild-type strain EC1 and the hfqEC1 deletion mutant were measured in Luria–Bertani (LB) medium (B) and minimal medium (MM) (C).
Microorganisms 10 01031 g001
Microorganisms 10 01031 g002 550
Figure 2. HfqEC1 regulates the production of cell wall–degrading enzymes. The wild-type strain EC1 of Dickeya oryzae and its derivatives were cultured in Luria–Bertani (LB) medium to an OD600 of 1.5. Production of Cel (A), Pel (B), and Prt (C) was measured in the media with their substrates and the corresponding methods. For semi-quantification of enzyme production, the radius of each halo was measured. Experiments were individually performed at least three times in triplicate. The data shown are the means ± SE (n = 3). ** p < 0.05; *** p < 0.01.
Figure 2. HfqEC1 regulates the production of cell wall–degrading enzymes. The wild-type strain EC1 of Dickeya oryzae and its derivatives were cultured in Luria–Bertani (LB) medium to an OD600 of 1.5. Production of Cel (A), Pel (B), and Prt (C) was measured in the media with their substrates and the corresponding methods. For semi-quantification of enzyme production, the radius of each halo was measured. Experiments were individually performed at least three times in triplicate. The data shown are the means ± SE (n = 3). ** p < 0.05; *** p < 0.01.
Microorganisms 10 01031 g002
Microorganisms 10 01031 g003 550
Figure 3. Disruption of hfqEC1 decreases bacterial swarming motility and biofilm formation. (A) Swarming motility of Dickeya oryzae EC1 and its derivatives. The diameters were measured after incubation at 28 °C for 18 h. (B) Swimming motility of D. oryzae EC1 and its derivatives. The plates were incubated at 28 °C for 24 h before photography. (C) Biofilm formation of D. oryzae EC1 and its derivatives. Bacterial strains were grown in super optimal broth plus glycerol (SOBG) medium at 28 °C with shaking for 18 h. Experiments were individually performed at least three times in triplicate. The data shown are the means ± SE (n = 3). ** p < 0.05.
Figure 3. Disruption of hfqEC1 decreases bacterial swarming motility and biofilm formation. (A) Swarming motility of Dickeya oryzae EC1 and its derivatives. The diameters were measured after incubation at 28 °C for 18 h. (B) Swimming motility of D. oryzae EC1 and its derivatives. The plates were incubated at 28 °C for 24 h before photography. (C) Biofilm formation of D. oryzae EC1 and its derivatives. Bacterial strains were grown in super optimal broth plus glycerol (SOBG) medium at 28 °C with shaking for 18 h. Experiments were individually performed at least three times in triplicate. The data shown are the means ± SE (n = 3). ** p < 0.05.
Microorganisms 10 01031 g003
Microorganisms 10 01031 g004 550
Figure 4. Expression of the zms cluster genes in Dickeya oryzae EC1 is regulated by HfqEC1. (A) Zeamine production assay. The filter-sterilized bacterial supernatants of D. oryzae EC1 and its derivatives were added to the wells in the bioassay plates, respectively. The bioassay plates were incubated at 37 °C for 24 h before photography. Experiments were repeated at least three times in triplicates. (B) QRT-PCR assay reveals the transcriptional regulation of key zms cluster genes by HfqEC1. (C) GFP promoter reporter fusion assay shows the regulation of zmsD gene by HfqEC1. Experiments were individually performed at least three times in triplicates. *** p < 0.01.
Figure 4. Expression of the zms cluster genes in Dickeya oryzae EC1 is regulated by HfqEC1. (A) Zeamine production assay. The filter-sterilized bacterial supernatants of D. oryzae EC1 and its derivatives were added to the wells in the bioassay plates, respectively. The bioassay plates were incubated at 37 °C for 24 h before photography. Experiments were repeated at least three times in triplicates. (B) QRT-PCR assay reveals the transcriptional regulation of key zms cluster genes by HfqEC1. (C) GFP promoter reporter fusion assay shows the regulation of zmsD gene by HfqEC1. Experiments were individually performed at least three times in triplicates. *** p < 0.01.
Microorganisms 10 01031 g004
Microorganisms 10 01031 g005 550
Figure 5. HfqEC1 is required for the virulence and hypersensitive response (HR) in Dickeya oryzae EC1. (A) Disease symptoms (left) and the rotting areas (right) on potato tubers inoculated with wild-type strain EC1, hfqEC1 mutant, and complementation strain of hfqEC1 mutant. Photograph was taken 2 d after inoculation. The numbers at the top of each column show the average rotting area ± SD from three repeats. ** p < 0.01. (B) Analysis of the capacity of D. oryzae EC1 in inhibiting rice seed germination. Rice seeds were treated with different bacterial dilutions as indicated and incubated at 28 °C for 7 days. Experiments were individually performed at least three times in triplicate. (C) The hypersensitive response induced by wild-type strain EC1, hfqEC1 mutant, and complementation strain of hfqEC1 mutant on leaves of Nicotiana benthamiana. An aliquot of 5 μL of bacterial cell cultures (OD600 = 0.05, 2.0 × 104 CFU/mL) was inoculated. The photograph was taken 24 h after infiltration.
Figure 5. HfqEC1 is required for the virulence and hypersensitive response (HR) in Dickeya oryzae EC1. (A) Disease symptoms (left) and the rotting areas (right) on potato tubers inoculated with wild-type strain EC1, hfqEC1 mutant, and complementation strain of hfqEC1 mutant. Photograph was taken 2 d after inoculation. The numbers at the top of each column show the average rotting area ± SD from three repeats. ** p < 0.01. (B) Analysis of the capacity of D. oryzae EC1 in inhibiting rice seed germination. Rice seeds were treated with different bacterial dilutions as indicated and incubated at 28 °C for 7 days. Experiments were individually performed at least three times in triplicate. (C) The hypersensitive response induced by wild-type strain EC1, hfqEC1 mutant, and complementation strain of hfqEC1 mutant on leaves of Nicotiana benthamiana. An aliquot of 5 μL of bacterial cell cultures (OD600 = 0.05, 2.0 × 104 CFU/mL) was inoculated. The photograph was taken 24 h after infiltration.
Microorganisms 10 01031 g005
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Shi, Z.; Wang, Q.; Wang, S.; Wang, C.; Zhang, L.-H.; Liang, Z. Hfq Is a Critical Modulator of Pathogenicity of Dickeya oryzae in Rice Seeds and Potato Tubers. Microorganisms 2022, 10, 1031. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10051031

AMA Style

Shi Z, Wang Q, Wang S, Wang C, Zhang L-H, Liang Z. Hfq Is a Critical Modulator of Pathogenicity of Dickeya oryzae in Rice Seeds and Potato Tubers. Microorganisms. 2022; 10(5):1031. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10051031

Chicago/Turabian Style

Shi, Zurong, Qingwei Wang, Shunchang Wang, Chengrun Wang, Lian-Hui Zhang, and Zhibin Liang. 2022. "Hfq Is a Critical Modulator of Pathogenicity of Dickeya oryzae in Rice Seeds and Potato Tubers" Microorganisms 10, no. 5: 1031. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms10051031

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop