The PafR Gene Is Required for Antifungal Activity of Strain MS 82 against Mycogone perniciosa

Bacterial strain MS82, isolated from the rhizosphere of a soybean plant, belongs to the species Pseudomonas fluorescens. The most important feature of strain MS82 is the production of antifungal activity against the mushroom pathogenic fungus Mycogone perniciosa but not against the mushroom fungus Agaricus bisporus. In this study, the mutant MS82MT19 generated with the EZ-Tn5 transposon system completely lost the antifungal activity against M. perniciosa. An open-reading frame named as PafR and predicted to code for a sensory box GGDEF/EAL domain protein, was disrupted in MS82MT19. To further confirm the function of this gene, site-directed mutagenesis with insertion of the terminatorless nptII cassette into the PafR gene was used to generate the mutant MS82SD19. As expected, there was no detectable antifungal activity of mutant MS82SD19 against M. perniciosa. These results suggest that the PafR gene plays an important role in the production of antifungal activity of P. fluorescens strain MS82.


Introduction
Mushrooms contain compounds such as minerals, vitamins, proteins and polysaccharides, and are therefore considered as nutritious foods [1] [2].Commercial mushroom production is around 16 million tons annually worldwide and more than 75% of these mushrooms are produced in China, where the total value of mushroom production is ranked the fifth in terms of crop value [3].Agaricus bisporus (Lange) Imbach, the white button mushroom, is grown mainly in North America, Europe, the Far East and Australasia, and is the predominant mushroom produced commercially worldwide [4].
The mushroom fungus A. bisporus is attacked by a number of fungal and bacterial pathogens that may cause significant production losses.The environmental conditions under which mushrooms are grown are conducive to the propagation of these pathogens and subsequent development of diseases in the mushroom crop.Mycogone perniciosa (Magnus) Delacroix is the fungal pathogen causing wet bubble disease on the mushroom A. bisporus in commercial production facilities worldwide [5].In recent years, the morbidity due to this disease was 40% and production was reduced more than 30% [6].Only a few fungicides, such as prochloraz-Mn and carbendazim, are registered for diseases control in commercial mushroom production [7].However, because of food safety issues, some of these fungicides are being phased out for mushroom production.Recently, the European community banned all benzimidazole-based fungicides such as carbendazim for control of the mushroom pathogens like M. perniciosa [8].Typically, biologically-based fungicides are ranked as no or low risk pesticides to animals and humans.Therefore, there is a demand for more biologically-based fungicides that may be used in mushroom disease management [9].
The bacterial genus Pseudomonas contains more than 100 species, many of which have been used for biocontrol of plant diseases [10].Strains of Pseudomonas spp.are frequently isolated from soils and many of them have been stu- died for their antimicrobial activities and ability to protect plants [11] [12] [13] [14].P. fluorescens lives in the soil and protects plant health by induction of plant defenses, nutrient cycling and antagonism against pathogens [15].It is important to understand the molecular mechanisms of biocontrol activity of bacterial strains used for the development of new antibiotics and biological fungicides.
Strain MS82 was preliminarily identified to P. fluorescens based on 16S rDNA analysis [16].The most important feature of strain MS82 is its antifungal activity against the white button mushroom fungal pathogen M. perniciosa, but not the mushroom fungus A. bisporus.This character indicates that MS82 has potential for development as a biopesticide for mushroom production.This study is to report the association of the PafR gene encoding a sensory box GGDEF/EAL domain protein with antifungal activity against the pathogenic fungus M. perniciosa.

Plasmids, Bacterial Strains and Culture Conditions
The plasmids and bacterial strains used in this study are listed in Table 1.Strain MS82 was isolated from the rhizosphere of a soybean plant grown at the R. R.  [21] with kanamycin (50 μg•mL −1 ) and tetracycline (25 μg•mL −1 ).

Multilocus Sequence Typing Analysis
Bacterial genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega Corporation.Madison, WI).Multilocus sequence typing (MLST) analysis was conducted as described by Jolley [22].The seven housekeeping genes (glnS, gyrB, ileS, nuoD, recA, rpoB and rpoD) were amplified from the DNA of strain MS82 using the primers listed in the website http://pubmlst.org/pfluorescens(Table 2).The cycling program included a 2 of MS82 was produced using MEGA6 software.

Bioassays for Antifungal Activity
The antifungal activities of Pseudomonas fluorescens MS82 and its mutants in this study were analyzed using PDA plate bioassays as described previously [23].Briefly, strain MS82 was grown overnight in NBY liquid medium, after which bacterial cells were collected by centrifugation and washed once with sterile distilled water.The cells were suspended in sterile distilled water to an optical density of 0.3 at 420 nm wavelength (approximately 2 × 10 8 CFU/mL), a drop of bacterial suspension (5 μL) was spotted on the center of each PDA plate, and plates were incubated 3 d at 28˚C.Each plate was then oversprayed with a standardized (OD 420 = 0. .The inhibitory zones of MS82 and its mutants against the indicator fungi were recorded and compared with each other.Three replicates of the plate bioassays were performed independently.

Random Mutagenesis Analysis
In order to find the genes associated with antifungal activity, EZ-Tn5 <R6Kγori/ KAN-2>Tnp Transposome Kit (Epicentre Biotechnologies, Madison, WI) was used as described by the manufacturer to randomly mutate strain MS82.Strain MS82 was grown in LB broth with shaking (220 rpm) at 28˚C until early log phase (OD 600 = 0.8 -1.0).The cells were harvested by centrifugation at 5000 rpm for 6 min at 4˚C, washed twice with the same volume and once with 0.5 volume of 300 mM sucrose, re-centrifuged, and finally re-suspended in 0.01 volumes of 300 mM sucrose [24].Before electroporation, the bacterial suspension was

DNA Cloning and Sequence Analysis of the Targeted Genes
Bacterial genomic DNA of mutant MS82MT19 was extracted as described above.
The bacterial genomic DNA was digested by restriction enzymes Pst I and Xba I, self-ligated, and then transformed into electrocompetent E. coli strain Trans-forMax EC100D pir + (Epicentre Biotechnologies, Madison, WI) as instructed by the kit to generate plasmids pMT19E1 and pMT19E2 (Table 1).Sequencing

Site-Directed Mutagenesis of the PafR Gene Targeted in Mutant MS82MT19 and Plasmid Construction for Complementation Assays
Efforts to complement the mutant MS82MT19 with a full-length targeted gene in trans were made through introduction of the plasmid pUCP26 harboring the full-length gene [18].However, the antifungal activity of the mutant could not be restored to the wild type level at all.Therefore, we used insertion of the nptII gene into the PafR gene to generate a nonpolar site-directed mutation.A 1kb fragment containing the SmaI site of the PafR gene of the wild-type strain MS82, for which PCR primers containing restriction sites of AatII and PstI were designed (FP-AatII-1: 5'-GAC AGA CGT CTA CCG AGG TCA ACC AGG TTG-3'; RP-PstI-1: 5'-CGA ACT GCA GCG AGG TCG AGT GCT CGA AAA C-3'), was cloned into the pGEM-T Easy vector to produce the plasmid pMS1.
The 1 kb fragment was digested by AatII and PstI and then subcloned into pBR325 [19], generating plasmid pMS2 containing tetracycline resistance.A 1.1-kb SmaI fragment of plasmid pBSL15 [20], which carries the nptII gene without a transcriptional terminator, was inserted into pMS2 at the SmaI site to generate plasmid pMS3 containing kanamycin and tetracycline resistance.Plasmid pMS3 was electroporated into strain MS82 for marker exchange mutagenesis as previously described [27], resulting in the generation of the mutant MS82SD19 which has resistance to kanamycin but sensitive to tetracycline.PCR was used to verify the double-crossover marker exchange in the mutant MS82SD19.The antifungal activity of the mutant MS82SD19 against M. perniciosa was evaluated with plate bioassays as described above.

Antifungal Activity of Strain MS82
Strain MS82 suppressed the mycelial growth of the fungal pathogen M. perniciosa with an inhibition zone of 34.17 ± 0.44 mm in the plate bioassay (Figure 2(B)).Strain MS82 also inhibited the growth of T. viride, and G. candidum (Figure 3).Interestingly, strain MS82 did not affect the mycelial growth of the mushroom fungus A. bisporus (Figure 2(A)).The antifungal activities of strain Pf0-1, the closest strain to MS82 based on phylogenetic analyses, were tested using the same methods as MS82.The results showed that strain Pf0-1 did not inhibit the growth of M. perniciosa, T. viride, or G. candidum (Figure 3).These results suggest that strain MS82 has an antifungal spectrum different from P. fluorescens strain Pf-01 and that its ability to inhibit a variety of fungi may be exploited for biocontrol and development of antibiotics for use in mushroom production.

Identification of the Mutant MS82MT19
Strain MS82MT19 was obtained from random mutagenesis that completely lost the antifungal activity and was chosen for further genetic analyses (Figure 2(C)).The 16S rRNA sequencing confirmed that t MS82MT19 was a mutant of strain MS82.Two plasmids, pMT19E1 and pMT19E2 (Table 1) carrying the re-

Sequence Analysis of the Targeted Gene in Mutant MS82MT19
Sequence analysis showed that the putative proteins encoded by the gene disrupted by the transposon in MS82MT19 shares high identities (99-100%) with sensory box GGDEF/EAL domain proteins of Pseudomonas.A 4.5kb flanking DNA region of the transposon, which was inserted in the genome of the mutant MS82MT19, was sequenced from the plasmids pMTE1 and pMTE2 (Table 1) by using primer walking and Sanger DNA sequencing.

Discussion
Pseudomonas strains can be isolated from various environmental niches such as soil and plant rhizospheres.P. fluorescens is a soil bacterium and many strains possess plant growth promoting functions [15].The genome sequences of Pseudomonas strains SBW25, Pf0-1 and Pf-5 revealed considerable diversity and their genomic heterogeneity is reminiscent of a species complex [28].Consequently, a few strains including Pf-5 were grouped as the new species P. protegens [29].The strain MS82, isolated from soybean rhizosphere, exhibits fluores- cence on Pseudomonas agar F plates under ultraviolet illumination.Phylogenetic analysis of MLST shows that MS82 is most close to P. fluorescens Pf0-1.Collectively the results of this study further confirmed that MS82 belongs to P. fluorescens.P. fluorescens not only has potential applications in biocontrol and bioremediation but also as a model for studying bacterial survival and fitness in soil [30].Notably, bioassays for antifungal activity reveled that MS82 has a unique antifungal spectrum that is different from P. fluorescens Pf0-1.This is not surprising due to the considerable diversity and genomic heterogeneity in the species [29], but it suggests that further study of the antifungal compounds produced by MS82 is needed.
The sensory box GGDEF/EAL domain is present in many organisms, including Vibrio, Pseudomonas, Staphylococcus, and is involved in regulation of cellular functions [31] [32] [33].The proteins with GGDEF and EAL domains are c-di-GMP cyclases or phosphdiesterase, respectively [34].At least one N terminal sensor domain such as GAF or PAS, which regulates activity by an irritant such as oxygen [35], is frequently contained in the GGDEF/EAL domain proteins [36].The pioneering work by Ross [37] showed the molecule c-di-GMP is an allosteric activator of cellulose synthase in Gluconacetobacter xylinus.
Another study showed that c-di-GMP was associated with production of cellulose in Agrobacterium tumefaciens [38].In other species, c-di-GMP levels affect transitions from sessile to moving cells and thereby play a role in the change between multicellular biofilm and planktonic motile cell status [34].The biofilm formation is also initiated by production of some compounds having antifungal activity, such as xantholysins, which are produced by the rhizosphere isolate P. putida BW11M1 [39].To the best of our knowledge, this is the first report of the association of a bacterial gene encoding sensory box GGDEF/EAL domain protein with production of antifungal activities.However, it remains to be determined how the PafR gene is involved molecularly in production of antifungal activity of strain MS82.
In this study, it was determined that the PafR gene of strain MS82 contains a GGDEF, an EAL and three PAS domains.Genetic analyses both random mutagenesis and site-directed insertional mutagenesis revealed the crucial role of the sensory box GGDEF/EAL-coding gene of MS82 in antifungal activity of that strain.Unexpectedly, we failed to restore the antifungal activity of the mutant MS82MT19 (PafR::miniTn5) by introduction of the full-length PafR gene and carried into the mutant by the Pseudomonas expression vector pUCP26 [17].However, nonpolar mutation using the terminatorless nptII cassette [40]

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conidial suspension of one of the three fungal indicator species: M. perniciosa JS01 (collection of Jindi Song), Trichoderma viride TX12 (Collection of Shien Lu) and G. candidum F-260 (a gift of Dennis Gross).The oversprayed plates were further incubated in the dark at 28˚C for 2 d.To evaluate the antifungal activity of MS82 against the mushroom fungus A. bisporus (Strain MS46, isolated from fresh mushroom), two PDA discs (5 mm in diameter) containing the mycelium of A. bisporus were placed 4 cm apart onto each of 9-cm PDA plates.A 5-μL aliquot of bacterial cell suspension (OD 420 = 0.3) was immediately spotted onto the same plate equidistant from the edge of each fungal disc.The inoculated plates were incubated in the dark for three weeks at 25˚C chilled on ice for 30 min.Aliquots of 80-μL cell suspension were mixed with one μL of EZ-Tn5 < R6Kγori/KAN-2 > Tnp Transposome and transferred to chilled 0.1 cm gap cuvettes.Electroporation was conducted using a Gene Pulser II (Bio-Rad Laboratories, Hercules, CA) with the settings of 25 μFD, 200 Ω and 1.8 kV.Immediately after electroporation, the cells were transferred into 3 ml LB broth and shaken (220 rpm) at 28˚C for 2h.The colonies growing on NBY plates supplemented with 50 μg•mL −1 kanamycin were picked up as potential mutants for antifungal plate bioassays.The mutant MS82MT19 that had no visible antifungal activity against M. perniciosa was cultured on NBY plates with 50 μg•mL −1 kanamycin for further study.The Tn5 transposon fragment was amplified by PCR with the primers EzTn5F (5'-TTA CGA AAC ACG GAA ACC-3') and EzTn5R (5'-TCT AAT ACC TGG AAT GCT GTT-3'), provided by the EZ-Tn5 <R6Kγori/KAN-2>Tnp Transposome Kit manufacturer, to confirm the presence of the transposon in the mutant.
reactions were performed using the primers KAN-2FP-1 and KAN-2RP-1 supplied in the transposon kit to the flanking genomic DNA fragments of the Tn5 transposon in mutant MS82MT19.The entire gene (i.e., the PafR gene) targeted by transposon in mutant MS82MT19 was sequenced by the Sanger primer walking method[25].In brief, five pairs of primers were designed and used to sequence the DNA flanking the Tn5 transposon in the plasmid pMT19E2.A consensus sequence of the entire gene region was generated using the Lasergene package.The gene functions were predicted through BLAST analysis against the GenBank database.The web-based software BPROM in the Softberry package was used for promoter prediction[26].

Figure 1 .
Figure 1.Phylogenetic tree of MS82 and related strains of Pseudomonas based on MLST analysis.

Figure 2 .
Figure 2. Plate bioassays of antifungal activities of P. fluorescens MS82 and its mutants against the fungal indictors M. perniciosa and A. bisporus.(A) The wild type strain MS82 (5 μl containing 1 × 10 6 cells) was inoculated onto the potato dextrose agar (PDA) plate simultaneously with A. bisporus (two 5 mm PDA discs with mycelia), then incubated in the dark for 3 weeks at 25˚C.(B)-(D) The wild type strain MS82 (B); MS82MT19 (C); and MS82SD19 (D) were each inoculated (5 μl l containing 1 × 10 6 cells) onto PDA plates and incubated in the dark for 3 d at 28˚C and then M. perniciosa was oversprayed on the plates.The oversprayed plates were further incubated in the dark at 28˚C for 2 d.

Figure 3 .
Figure 3. Plate bioassays for antifungal activities of P. fluorescens strains MS82 and Pf0-1.One droplet of bacterial suspension (5 µl containing ~10 6 cells) was inoculated onto each potato dextrose agar plate.Plates (A) (B) and (C) were inoculated with MS82; plates (D) (E) and (F) were inoculated with Pf0-1.The plates were incubated in the dark for 2 d at 28˚C before being oversprayed with spore suspension of M. perniciosa (A) (D); T. viride (B) (E) or G. candidum (C) (F); then incubated another 3 d in the dark at 28˚C.

3 . 5 .
Further analysis revealed that only one open-reading frame of 3849 nt encoding the sensory box GGDEF/ EAL domain protein is disrupted by the transposon in MS82MT19.This gene was named as the PafR gene (Pseudomonas antifungal regulatory gene) and its sequence was deposited into GenBank with the accession number KU510320.The promoter region (−35 box: ATGCTA and −10 box: GCTTATAGT) was identified 38 nucleotides upstream of the putative start codon of the predicted protein PafR.A typical sequence-dependent terminator was not found from the untranslational 3' end of the gene.The BLAST results revealed that the putative protein of this gene shares 99% amino acid identity to the sensory box GGDEF/ EAL domain protein of P. fluorescens Pf0-1 (GenBank accession: ABA76613).In addition, the results showed that the transposon in the MS82MT19 genome was inserted between the nucleotides 1393 and 1394 of the sensory box GGDEF/ EAL domain gene.Characterization of the Role of the Sensory box GGDEF/EAL Domain Gene in Production of the Antifungal Product The role of sensory box GGDEF/EAL domain PafR gene targeted in MS82MT19 in production of antifungal activity was further determined using the site-directed mutagenesis.After 16S RNA DNA sequencing confirmed the identity of the mutant MS82SD19, the insertion of the terminitorless nptII cassette in the PafR gene was determined by PCR analysis with the primers FP-AatII-1 and RP-PstI-1.As expected, the PCR products were 1.1 kb in size from genomic DNA of the wild type strain MS82 and 2.2 kb containing the nptII cassette from both MS82SD19 genomic DNA and plasmid pMS3 (Figure 4), respectively.The

Figure 4 .
Figure 4. PCR confirmation of site-directed insertional mutation in the mutant MS82SD19.The primers FP-AatII-1 and RP-PstI-1 were used to amplify the fragment of the PafR gene.Lane 1: PCR product of the partial PafR gene amplified from MS82 (1.1 kb); Lane 2: PCR product of the partial PafR gene amplified from plasmid pMS3 (2.2 kb); Lane 3: PCR product of the partial PafR gene amplified from mutant MS82SD19 (2.2 kb); Lane M: 1kb ladder.
[41] verified the role of the PafR gene in production of antifungal activity.This indicates that the function of the PafR gene may also be affected by the other factors, such as expression level of the gene[42] [43].Nevertheless, the results of this research indicate the PafR gene encoding the sensory box GGDEF/EAL domain plays an important role in antifungal activity and provides a crucial clue to understand molecular mechanisms of antifungal activity of MS82.Further work is under way to identify the genes required for production of the antifungal agents of MS82.The studies will provide useful clues for development of MS82 as a biological control agent to manage fungal diseases of mushroom cultivation.