pathogens Article
Association and Expression of Virulence from Plasmids of the Group B Strain in Pseudomonas syringae pv. eriobotryae Tran Dang Khanh 1 and Tran Dang Xuan 2, * 1 2
*
ID
Agricultural Genetics Institute, Pham Van Dong Street, Hanoi 122300, Vietnam;
[email protected] Division of Development Technology, Graduate School for International Development and Cooperation (IDEC), Hiroshima University, Higashi Hiroshima 739-8529, Japan Correspondence:
[email protected]; Tel./Fax: +81-82-424-6927
Received: 5 February 2018; Accepted: 12 April 2018; Published: 14 April 2018
Abstract: Pseudomonas syringae pv. eriobotryae causes serious stem canker in loquat (Eriobotrya japonica) trees. This study was conducted to determine whether plasmids are involved with its virulence. The strain NAE89, which belonged to the B group, harbored two plasmids at approximately 6.2 and 50 Mdal that caused stem canker and halo leaf spots on loquat plants. Following digestion with BamHI and ligation into the BamHI cloning site of the broad range host cosmid pLAFR3, four DNA fragments at 3.8, 6.6, 12.3, and 22.8 kb were generated. Although the plasmid-encoded virulence gene psvA was undigested with the BamHI, the halo leaf spot gene may be adjacent to the psvA gene was digested. A pLAFR3 cosmid clone was introduced into the non-pathogenic PE0 and NAE89-1 strains by triparental matings and the pathogenicity was recovered. As a result, the pLAFR3 cosmid clone was introduced into the largest size DNA fragment of 22.8 kb and determined to be the causal agent of canker on the stem of the loquat. This study revealed that the psvA gene, previously found in the 50 Mdal plasmid, was also observed in the 22.8 kb DNA fragment. Keywords: Pseudomonas syringae pv. eriobotryae; pigment; pathogenic; psvA; virulence gene
1. Introduction Pseudomonas syringae pv. eriobotryae is a pathogenic bacterium that causes stem canker in loquat trees. The pathogen attacks all plant parts, causing weak growth and reduction of commercial value of fruits [1,2]. Damage from this disease has been documented in Argentina, Australia, China, Japan, New Zealand, and the US [3–9]. The stem canker bacterium has been classified into three groups—A, B, and C—based on their pigment production and pathogenicity to loquat leaves [10]. The A and B strains do not produce pigment. Unlike group A strains, group B strains are pathogenic to mesophyll. The group C strains produce dark brown pigment, but are not pathogenic to mesophyll [11,12]. However, all of the three bacterium groups cause pathogenicity to the stems of loquat plants. In particular, the group B strains cause both stem canker and halo leaf spot. The strain NAE6 was found to belong to the A group of Pseudomonas syringae pv. eriobotryae (Ps. pv. eriobotryae). While it contained the 25, 52, and 60 Mdal plasmids, the 52 Mdal plasmid appeared to be required for virulence [13]. A pLAFR3 cosmid clone, which contained a 23 kb inserted DNA derived from the 52 Mdal plasmid, was designated the pVIR6 and reintroduced into a cured virulent PE0 strain with the helper plasmid pRK2014 [13]. The conjugates that received pVKR6 regained virulence. It was proposed that the 52 Mdal plasmid possessed the virulence gene(s) [13]. In the group A strains, a loss of the 85 Mdal plasmid was associated with the disappearance of virulence. Attempts to reintroduce the 85 Mdal plasmid into a cured strain have not been successful [13].
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Several studies were conducted to determine the involvement and expression of plasmids with regards to virulence gene(s) such as the halo bright pathogen of bean, Pseudomonas savastanoi pv. phaseolicola, which consists of nine races, eight of which harbored large plasmids of about 150 kb. The pAV511 resulted in the loss of virulence in bean cultivars [14]. A similar native plasmid for curing of an approximately 82 kb native plasmid by heat shock (32 ◦ C) from a strain of Ps. syringae pv. eriobotryae, the causal agent of stem canker in the loquat, led to a failure of pathogenicity in the host plant [13]. A single gene, psvA, identified by Hiehata et al. [15] and named Pse-a, was capable of restoring pathogenicity [13,15]. The gene, with a low % G + C and potential hrpL promoter sequence, showed no similarity to other genes in the databases. An exception was a small portion of the N-terminus of the putative protein products, which had a similarity to AvrA [16]. Many plant pathogens, particularly the soft-rot pathogen Erwinia carotovora and some pathovars of Xanthomonas campestris, secreted extracellular enzymes that were specific to chromosomal genes [17]. The others included the pathogen Burkholderia cepacia and some plant strains cause bulb-rot of the onion (Allium cepa) [17]. The production of an Endopolygalacturonase strain was found to be due to the gene, pehA, which was located on a 200 kb plasmid [18]. A potential vir gene, designated virPphA, restored virulence to bean when introduced into the cured race 7 strain RW60 [18]. However, it conferred a virulence to certain soybean (Glycine max) cultivars. Given the common regulatory features of virPphA and the avr genes in Ps. syringae, implied that they might be the virulence genes [18]. A gene showing dual avr/vir gene behavior was isolated from the pPATH plasmid of Erwinia herbicola pv. gypsophilae [19]. It was reported that the resistance to loquat canker (group A) controlled by a single dominant gene, designated as Pse-a [15]. The wild species bronze loquat (Eriobotrya deflexa) and was mapped [12] to reveal the presence of Pse-a, with the constructed linkage group spanned by 69.4 cM and had an average marker density of 2.6 cM [12]. Resistance to the group C bacteria was controlled by a recessive gene Pse-c [20], and mapped [2]. Hiehata et al. [21] screened 52 loquat cultivars for resistance to groups A, B, and C. There were 25 resistant cultivars, whereas 22 were susceptible to group A. Most of these observations exhibited similar responses to group B, thus it was suggested that the resistance of the gene Pse-a might be associated with groups A and B [21]. However, further pathogenic analyses on group B have not been carried out. Although much attention has been focused on the A and C strain groups of the Ps. pv. eriobotryae as mentioned above, little information was available on the B strain group, which uniquely caused both stem canker and halo leaf spot (Supplement 1). In addition, the involvement of the pathogenic gene(s) involved in the B strain groups has remained unknown. Hence, this study was conducted to investigate the pathogenic expression of the plasmids in the B strain group. 2. Results 2.1. Cloning Fragments from Plasmid NAE89 Plasmids of the NAE89 strain were extracted following a method described in Kado and Liu [22], and used for agarose gel electrophoresis. Similar plasmid groups were extracted, including the NAE6 bacteria (A group) and NAE57 bacteria (C group). The NAE89 strains harbored two kinds of plasmids at 6.2 Mdal and 50 Mdal (Figure 1A). It was known that these strains belong to a group with similar plasmid patterns and had a high possibility of being borne by the plasmid [13]. It was assumed that the pathogenic gene, psvA associated with the establishment of canker disease, and the gene(s) involved in halo leaf spot formation, implicated that the plasmids of the NAE89 strain existed. Although the psvA gene was not cut by BamHI, the plasmids of the NAE89 strain were extracted for the first time, cut by BamHI, and used for agarose gel electrophoresis.
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Figure 1. (A) Agarose gel electrophoresis of the plasmids from Ps. pv. eriobotryae; Lanes 1: 60, 52, and 25 Mdal plasmids of NAE6 (group A); Lanes: 50, 6.2 Mdal plasmids of NAE89 (group B); Lanes 3: 85, 39, 32 plasmids of NAE57 (group C); (B) Agarose gel electrophoresis of NAE89 plasmids digested with BamHI. Lane 1: λ/HindIII, 23.1, 9.4, 6.5, 2.3, and 2.0 kb. Lane 2: NAE89/BamHI with 22.8 (F1), 12.3 (F2), 6.6 (F3), and 3.8 kb (F4). (A)Agarose Agarose gel electrophoresis ofthe the plasmids from pv. eriobotryae; Lanes 60, 52, Figure (A) electrophoresis plasmids from Ps.Ps. pv. eriobotryae; Lanes 1: 60,1:52, It was 1.assumed thatgel the pathogenic ofgene, psvA associated with the establishment ofand canker and 25 Mdal plasmids of NAE6 (group A); Lanes: 50, 6.2 Mdal plasmids of NAE89 (group B); Lanes 3: the 25 Mdal of NAE6 (groupinA); Lanes: 6.2 Mdal plasmids of NAE89 (group B);plasmids Lanes 3: 85, disease, and plasmids the gene(s) involved halo leaf50,spot formation, implicated that the of 85, 39, 32 plasmids of NAE57 (group C); (B) Agarose gel electrophoresis of NAE89 plasmids digested 39, strain 32 plasmids of NAE57 (group (B) gene Agarose electrophoresis of NAE89 plasmidsof the NAE89 NAE89 existed. Although theC); psvA wasgelnot cut by BamHI, the plasmids with BamHI. Lane 1: λ/HindIII, 23.1, 9.4, 6.5, 2.3, and 2.0 kb. Lane 2: NAE89/BamHI with 22.8 22.8 (F1), (F1), 12.3 12.3 with BamHI. Lane 1: λ/HindIII, 23.1, 9.4, 6.5, 2.3, and 2.0 kb. Lane 2: NAE89/BamHI with strain were extracted for the first time, cut by BamHI, and used for agarose gel electrophoresis. (F2), 6.6 (F3), and 3.8 kb (F4). (F2), 6.6 (F3), and 3.8 kb (F4). Consequently, four DNA fragments—22.8 kb (F1), 12.3 kb (F2), 6.6 kb (F3), and 3.8 kb (F4)— were produced (Figure 1B). The fragment of λ/HindIII was used as a standard and its molecular It was that thesize pathogenic gene, psvA associated with the establishment of canker Consequently, four DNA fragments—22.8 kbfragment. (F1), 12.3 kb (F2), 6.6 kb (F3), 3.8packaging, kb (F4)—were weight usedassumed to calculate the of each DNA After ligation via inand vitro the disease, and the gene(s) involved in halo leaf spot formation, implicated that the plasmids of producedof (Figure 1B). The fragment λ/HindIII used with as a standard andintroduced its molecular plasmids the pLAFR3 and NAE89of strains were was digested BamHI and intoweight thethe E. NAE89 strain existed. Although the psvA gene was not cut by BamHI, the plasmids of the NAE89 used to calculate the size of each DNA fragment. After ligation via in vitro packaging, the plasmids of coli strain DH5. All plasmids of the 200 obtained clones were examined (data not shown) based on strain were extracted for the first time, cut by BamHI, and used for agarose gel electrophoresis. the pLAFR3 and NAE89 strains were digested with BamHI and introduced into the E. coli strain DH5. their patterns and molecular weights. Their sizes became larger than pPLAFR3 in each plasmid, and Consequently, four DNA were fragments—22.8 kb (F1),2). 12.3 kb shown) (F2), 6.6the kb plasmids (F3), and derived 3.8 kb (F4)— All of the 200 obtained clones were examined (data not based on their patterns and the plasmids cloned pLAFR3 plasmids identified (Figure Furthermore, from were produced (Figure 1B). The fragment of λ/HindIII was used as a standard and its molecular molecular weights. Their sizes became larger than pPLAFR3 in each plasmid, and the cloned pLAFR3 those clones were digested with BamHI and agarose gel electrophoresis was performed. weight used towere calculate the size on of DNA fragment. After fragments ligation viaof inthe vitro packaging, the plasmids were identified (Figure 2).each Furthermore, theBamHI plasmids derived from those clones were Investigations undertaken the clones of the observed NAE89 plasmids of the pLAFR3 and NAE89 strains were digested with BamHI and introduced into the E. digested with BamHI and agarose gel electrophoresis was performed. Investigations were undertaken plasmid. coli strain DH5. All BamHI plasmids of the 200 obtained clones wereplasmid. examined (data not shown) based on on the clones of the fragments of the observed NAE89 their patterns and molecular weights. Their sizes became larger than pPLAFR3 in each plasmid, and the cloned pLAFR3 plasmids were identified (Figure 2). Furthermore, the plasmids derived from those clones were digested with BamHI and agarose gel electrophoresis was performed. Investigations were undertaken on the clones of the BamHI fragments of the observed NAE89 plasmid.
Figure 2. Size of DNA fragment of the pLAFR3 cosmid clone. Lane 1: NEA89 plasmid/BamHI Figure 2. Size of DNA fragment of the pLAFR3 cosmid clone. Lane 1: NEA89 plasmid/BamHI (F1–F4); (F1–F4); Lane 2: pLAFC1/BamHI (F1); Lane 3: pLAFC2/BamHI (F1); Lane 4: pLAFC3/BamHI (F2); Lane 2: pLAFC1/BamHI (F1); Lane 3: pLAFC2/BamHI (F1); Lane 4: pLAFC3/BamHI (F2); Lane 5: Lane 5: pLAFC4/BamHI (F2); Lane 6: pLAFC5/BamHI (F2); Lane 7: pLAFC6/BamHI (F2); Lane 8: pLAFC4/BamHI (F2); Lane 6: pLAFC5/BamHI (F2); Lane 7: pLAFC6/BamHI (F2); Lane 8: pLAFC7/BamHI (F2, F4); Lane 9: pLAFC8/BamHI (F2, F3); Lane 10: pLAFC9/BamHI (F1); Lane 11: pLAFC7/BamHI (F2, F4); Lane 9: pLAFC8/BamHI (F2, F3); Lane 10: pLAFC9/BamHI (F1); Lane 11: standard of λ/HindIII. standard of λ/HindIII.
Results2.showed that fragment pLAFC1,ofpLAFC2, andcosmid pLAFC9 were inserted intoplasmid/BamHI the F1 fragment at 22.8 kb. Figure Size of DNA the pLAFR3 clone. Lane 1: NEA89 (F1–F4); Whilst pLAFC3, pLAFC4, pLAFC5, and3:pLAFC6 were inserted into the F2 fragment at(F2); 12.3 kb, pLAFC8 Lane 2: pLAFC1/BamHI (F1); Lane pLAFC2/BamHI (F1); Lane 4: pLAFC3/BamHI Lane 5: pLAFC4/BamHI Lane 6: pLAFC5/BamHI Lane 7: into pLAFC6/BamHI Lane 8:and a was inserted into the(F2); F2 and F3 fragments, pLAFC7 (F2); was inserted the F2 and F4(F2); fragments, pLAFC7/BamHI (F2, inserted F4); Laneinto 9: pLAFC8/BamHI F3); Lane (Figure 10: pLAFC9/BamHI (F1); although Lane 11: the fragment of 9.4 kb was the BamHI site(F2, of pLAFR3 2). In addition, standard of kb λ/HindIII. fragment of 9.4 was also newly inserted into pLAFC7, the F3 and F4 fragments were joined together.
Results showed that pLAFC1, pLAFC2, and pLAFC9 were inserted into the F1 fragment at 22.8 kb. Whilst pLAFC3, pLAFC4, pLAFC5, and pLAFC6 were inserted into the F2 fragment at 12.3 kb, pLAFC8 was inserted into the F2 and F3 fragments, pLAFC7 was inserted into the F2 and F4 fragments, and a fragment of 9.4 kb was inserted into the BamHI site of pLAFR3 (Figure 2). In Pathogens 2018, 7, 41 4 of 8 addition, although the fragment of 9.4 kb was also newly inserted into pLAFC7, the F3 and F4 fragments were joined together. FourNAE89 NAE89plasmid plasmid fragments digested BamHI were inserted. and NAE89-1, Four fragments digested withwith BamHI were inserted. PE0 andPE0 NAE89-1, a bacteriala bacterial strain non-pathogenic to the loquat, were introduced into NAE89 via triparental matings. strain non-pathogenic to the loquat, were introduced into NAE89 via triparental matings. Consequently, Consequently, inserted caused fragment sizes In become smaller. In the the inserted PE0the caused one PE0 deletion, andone all deletion, fragmentand sizesallbecome smaller. the case of NAE89-1, case of NAE89-1, the deletion occurred in two strains, while most were completely introduced. The the deletion occurred in two strains, while most were completely introduced. The result of a deletion result of a deletion varied between pLAFRC1 and pLAFRC2, in longest which F1 was theinserted longest fragment varied between pLAFRC1 and pLAFRC2, in which F1 was the fragment into PE0 inserted into PE0 (data not shown). (data not shown). 2.2. Pathogenic Pathogenic Experiment Experiment 2.2. The PE0 PE0 bacteria bacteria of of the the loquat loquat did did not not carry carry the the code code for for the the psvA psvAgene. gene. As As aa result, result, the the PE0 PE0 The bacteriadid didnot notexhibit exhibitcanker cankersymptoms. symptoms.Therefore, Therefore,the theNAE89 NAE89was wasa amutation mutationstock stock that expressed bacteria that expressed a a loss of halo symptoms during its formation. The PE0 and NAE89-1 were introduced into loss of halo leaf leaf symptoms during its formation. The PE0 and NAE89-1 were introduced into pLAFR3, pLAFR3, in which each BamHI of fragment of the NAE89was plasmid wasby inserted by triparental matings. in which each BamHI fragment the NAE89 plasmid inserted triparental matings. pLAFC1 pLAFC1 andfrom pLAFC2 the F1 fragment with largest size and were inserted, and showed and pLAFC2 the F1from fragment with the largest sizethe were inserted, showed pathogenicity in pathogenicity in Other the loquat stem. Other inserted fragments (F2–F4)pathogenicity did not display in the loquat stem. inserted fragments (F2–F4) did not display in pathogenicity the loquat stem the loquat stem (Figure 3A,B). (Figure 3A,B).
Figure Figure 3.3. Pathogenic Pathogenic expression expression of of each each cloned cloned plasmid plasmid inoculated inoculated to to loquat loquat stem. stem. (A) (A) Virulent Virulent expression: expression: 1. 1. PE0-TM PE0-TM (C2); (C2); 2. 2. PE0-TM PE0-TM (C5); (C5); 3. 3. PE0-TM PE0-TM (C8); (C8); 4. 4. NAE6; NAE6; 5. 5. PE0-TM PE0-TM (C7); (C7); (B) (B) Invirulent Invirulent expression expression(controls): (controls): 1. 1. PE0-TM PE0-TM (C1) (C1) (PE0 (PE0 without withoutinsertion), insertion),(2) (2)distilled distilledwater; water;TM: TM:treatment. treatment.
2.3. Southern Southern Hybridization Hybridization 2.3. Thepathogenic pathogenic examination revealed that the F1 fragment of size the showed largest pathogenicity. size showed The examination revealed that the F1 fragment of the largest pathogenicity. To investigate whether gene the pathogenic gene psvA existed, a Southern hybridization To investigate whether the pathogenic psvA existed, a Southern hybridization was performed was performed usingpsvA the pathogenic psvA (canker bacteria the loquat) as a probe. Consequently, using the pathogenic (canker bacteria of the loquat) as of a probe. Consequently, the F1 fragment the F1 fragment the largest size was andofthe of thebecame psvA gene(s) hybridized with hybridized the largest with size was formed, and theformed, existence theexistence psvA gene(s) clear became4B, clear (Figure lane 1).the In50 addition, the 50 Mdal the NAE89 strain were and aformed. hybrid (Figure lane 1). In 4B, addition, Mdal plasmid of theplasmid NAE89 of strain and a hybrid wereF1formed. Theof F1the fragment the NAE89had obviously had aorigin 50 Mdal origin (Figure 4A, Of lanewhich, 2). Of The fragment NAE89ofobviously a 50 Mdal (Figure 4A, lane 2). which, the 50band Mdalof band the plasmid (unclear), hybrid was formedininthe theposition position the 50 Mdal theof plasmid was was thin thin (unclear), andand thethe hybrid was formed of chromosomal chromosomalDNA. DNA.This Thiswas was part part of of the the closed closed circular circular 50 50 Mdal fractured fractured plasmid, plasmid, and and considered considered of at aa similar similar position positionas asthe the linear linearchromosome chromosomeusing usingthe theagarose agarosegel gelelectrophoresis electrophoresismethod. method. at However, the NAE6 strain was the 52 Mdal plasmid, from which the existence of the psvA gene was identified and hybrids were formed (Figure 4, lane 3). The pathogenicity was exhibited through a partial deletion when pLAFC1 and pLAFC2 were introduced into PE0 by triparental matings. It was proposed that there were deleted parts other than caused by the psvA gene(s), and the deletion
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However, However, the the NAE6 NAE6 strain strain was was the the 52 52 Mdal Mdal plasmid, plasmid, from from which which the the existence existence of of the the psvA psvA gene gene was was identified identified and and hybrids hybrids were were formed formed (Figure (Figure 4, 4, lane lane 3). 3). The The pathogenicity pathogenicity was was exhibited exhibited through through 2018, 7, 41 when pLAFC1 and pLAFC2 were introduced into PE0 by triparental matings. 5 of 8 aaPathogens partial partial deletion deletion when pLAFC1 and pLAFC2 were introduced into PE0 by triparental matings. It It was was proposed proposed that that there there were were deleted deleted parts parts other other than than caused caused by by the the psvA psvA gene(s), gene(s), and and the the deletion deletion plasmids plasmids introduced into PE0 were investigated by Southern hybridization. Each of these plasmids plasmids introduced introduced into into PE0 PE0 were were investigated investigated by by Southern Southern hybridization. hybridization. Each Each of of these these plasmids plasmids formed a hybrid with psvA, which indicated the existence of the psvA (Figure 5). formed of the the psvA psvA (Figure (Figure 5). 5). formed aa hybrid hybrid with with psvA, psvA, which which indicated indicated the the existence existence of
Figure 4. Southern hybridization NAE89 plasmid P. the of of thethe NAE89 plasmid from from the P. the syringae pathovarpathovar using theusing virulence Figure 4. 4. Southern Southernhybridization hybridization of the NAE89 plasmid from the P. syringae syringae pathovar using the virulence as aa probe. (A) gel. 1: was with gene psvAgene as apsvA probe. Ethidium bromidebromide gel. Lane 1: Lane NAE89 was digested with BamHI, lane 2: virulence gene psvA as (A) probe. (A) Ethidium Ethidium bromide gel. Lane 1: NAE89 NAE89 was digested digested with BamHI, BamHI, lane 2: 3: lane 4: aa standard; Southern blot gel panel. NAE89, lane 3:lane NAE6, lane 4: λ/HindIII as aas (B)(B) Southern blot F1 lane 2: NAE89, NAE89, lane 3: NAE6, NAE6, lane 4: λ/HindIII λ/HindIII asstandard; standard; (B) Southern blotofof ofgel gelinin inpanel. panel. The The F1 F1 fragment largest size was formed from hybridization. fragment with with the the largest size was formed from hybridization.
Figure Southern hybridization showing that pathogenicity recovered Figure 5. that pathogenicity waswas recovered by a by non-pathogenic strain 5. Southern Southernhybridization hybridizationshowing showing that pathogenicity was recovered by aa non-pathogenic non-pathogenic strain in the triparental mating trial; (A) 1% agarose gel; (B) Southern blot of gel; Lane 1: NAE6; Lane in the in triparental mating trial; trial; (A) 1% gel; gel; (B) Southern blotblot of gel; Lane 1: NAE6; Lane 2: strain the triparental mating (A)agarose 1% agarose (B) Southern of gel; Lane 1: NAE6; Lane 2: PE0-TM (C2) Lane PE0-TM (C1) Lane PE0-TM (C1) b; psvA PE0-TM (C2) a;a; Lane (treatment). 2: PE0-TM (C2) a; Lane3:3: 3:PE0-TM PE0-TM(C1) (C1)a;a; a;Lane Lane4:4: 4:PE0-TM PE0-TM(C1) (C1)b; b; psvA psvA used as aa probe; probe; TM TM (treatment). (treatment).
3. 3. Discussion Discussion Discussion This showed that the strains group A three plasmids 25, 52, 60 thethe group A harbored three plasmids at 25,at Mdal; This study study showed showedthat thatthe thestrains strainsofof of the group A harbored harbored three plasmids at52, 25,and 52,60and and 60 Mdal; the group B strains were at 6.2 and 50 Mdal; and the group C strains were at 32, 39, and 85 the group B strains were at 6.2 and Mdal; and the group C strains were at 32, at 39,32, and Mdal Mdal; the group B strains were at 6.250and 50 Mdal; and the group C strains were 39,85and 85 Mdal (Figure 1A). The study that psvA gene in 50 of (Figure 1A). The present study showed that the psvA gene existed the 50 Mdal of plasmid the NAE89 Mdal (Figure 1A). The present present study showed showed that the the psvA geneinexisted existed in the theplasmid 50 Mdal Mdal plasmid of the NAE89 strain, which involved in stem canker formation. An attempt was made to detect strain, which involved in stem canker formation. An attempt was made to detect a phenotypic marker the NAE89 strain, which involved in stem canker formation. An attempt was made to detect aa phenotypic marker encoded on Mdal in By study, that was encoded onthat thewas 52 Mdal plasmid in a52 manner [23]. By this manner study, it [23]. was found the phenotypic marker that was encoded on the the 52similar Mdal plasmid plasmid in aa similar similar manner [23]. By this thisthat study, it was that the B bacterium caused canker halo leaf spots of group B strain bacterium caused stem canker and halostem leaf spots of and loquat trees harbored 6.2trees and it was found found that the group group B strain strain bacterium caused stem canker and halo leafand spots of loquat loquat trees and harbored and 50 Mdal gene 6.2 fragments. and harbored 6.2 and 50 50 Mdal Mdal gene gene fragments. fragments. Kamiunten [16] reported that the pLAFR3 cosmid clone, pVIR6, which contained aa virulence cosmid clone, pVIR6, which contained a virulence gene Kamiunten [16] [16] reported reportedthat thatthe thepLAFR3 pLAFR3 cosmid clone, pVIR6, which contained virulence gene 23 insert DNA region. This gene from the 52 plasmid in thein 23the kb insert virulence gene originated from the 52 Mdal plasmid fragment of gene in the 23 kb kbDNA insertregion. DNA This region. This virulence virulence gene originated originated from the 52 Mdal Mdal plasmid fragment of the group A strain Ps. syringae pv. eriobotryae. The deletion analyses of pVIR6 determined the groupofAthe strain Ps. A syringae pv.syringae eriobotryae. The deletion analyses ofanalyses pVIR6 determined that about fragment group strain Ps. pv. eriobotryae. The deletion of pVIR6 determined
7 kb of the inserted DNA restored pathogenicity to an avirulent PE0 strain. The plasmid in which the deletion occurred was denoted to be the pKPN35. A 6961 bp inserted to the DNA of pKPN35 was sequenced, and four possible open reading frames were found in tandem [16]. Of which, ORF1 and
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ORF4 exerted no significant homology to known genes. The ORF2 had an amino acid sequence similar to the transposase of the IS5 of E. coli. A recombinant plasmid pNSF1 containing only the ORF3 region restored pathogenicity to the avirulent PE0 strain. However, an ORF3 mutant of pNSF1, which was constructed by deleting a 580 bp BssHII segment from ORF3, failed to restore virulence. Consequently, the ORF3 was identified as a virulent psvA gene [16]. In this experiment, it was found that the BamHI fragment of the plasmid NAE89 strain, belonging to group B contained a known psvA virulence gene. The BamHI cutting site and the halo leaf spot gene(s) being adjacent to the psvA gene of the cosmid vector pLAFR3 were also examined. Furthermore, the NAE89 strain plasmid produced four fragments (F1 fragment, 22.8 kb; F2 fragment, 12.3 kb; F3 fragment, 6.6 kb; F4 fragment, 3.8 kb). Following the result of investigating among 200 clones (data not shown), it was possible to obtain the E. coli DH5 into the four digested BamHI fragments. Hence, each of BamHI fragments (F1–F4) was introduced into NAE89-1 via triparental matings with PE0. When being inoculated (Figure 3), the F1 fragment of the largest size showed causal canker symptoms on the stems. This study was the first to provide evidence that the psvA gene associated with the 50 Mdal plasmid, and proposed that the 50 Mdal plasmid of the group B strain might play an important role in the development of canker symptoms on loquat stems and pathogenic activities. These findings provide useful information for the clarification of the halo leaf spot gene(s). Further trials with sequences should be carried out to re-estimate the positions of the observed fragments (F1–F4). In addition, the involvement of the psvA gene in the group B strain should be further investigated. 4. Materials and Methods 4.1. Bacterial Strains, Plasmids, Culture Conditions, and Chemicals The bacterial strains and studied plasmids were listed in Table 1. The Pseudomonas syringae pv. eriobotryae strains were kindly supplied by Nagasaki Fruit-Tree Experiment Station, Nagasaki Prefecture, Japan. They were originally isolated from loquat stem cankers collected from various locations in Japan, and used as a probe in this study. The bacteria were ordinarily cultured in yeast extract–peptone (YP) medium (yeast extract 5 g, polypetone 10 g, NaCl 5 g, glucose 1 g, 1000 mL distilled water, pH 7.0) at 25 ◦ C, following a method described in Hasebe et al. [24]. For growth on solid medium, 1.5% agar was added. Escherichia coli strains were grown in similar medium at 37 ◦ C. HindIII-digested λ DNA was used as markers of standard molecular weight. Table 1. Bacterial strains and plasmids Strains or Plasmids Pseudomonas syringae pv. eriobotryae NAE6 PE0 NAE89 NAE89-1 NAE57 E. coli DH5 pET-3a (psvA) pRK2013 pLAFR3
Relevant Properties
Wild-type virulence in group A Plasmid lost NAE 6 Wild-type virulence in group B The variant of lost halo gene formation Wild-type in the C group E. coli bacteria and bacterium strain Vector inserted with BamHI to psvA used as a probe in this study, isolated from a bacteria lesion of the strain group A Helper plasmid Cosmid vector
Source or Reference Nagasaki Fruit-Tree Experiment Station, Japan [13] [13] Nippongene Nippongene Nippongene Nippongene Stratagene This study This study
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4.2. Plant Inoculation Young yellow-green loquats were selected and washed in 70% ethanol to disinfect them of bacteria or fungi. Bacteria grown on YP agar (1.5%) medium for 24 h were inoculated into one-year-old loquat stems using a needle. The inoculated plants were placed in polyethylene bags for 12 h to keep the humidity high, then they were transferred to a growth chamber at 25 ◦ C with a 12 h photoperiod. The symptom of halo spot formation developments on the stems was observed at 60 days after inoculation. 4.3. General DNA Manipulation and Clone Plasmid Plasmid extraction, restriction enzyme digestion, DNA ligation, plasmid clone, and agarose gel electrophoresis were carried out using standard procedures [22,25]. Plasmids were introduced into the E. coli by transformation and into Ps. syringae pv. eriobotryae by triparental matings with pRK2013 as a conjugative plasmid. 4.4. Southern Hybridization Experiments The isolation of total genomic DNA was performed following a method previously described by Kamiunten [13]. The cosmid vector pLAFR3 was digested with BamHI to convert to a single linear fragment and electrophoresed. The linearized pLAFR3 was then isolated from the agarose gel with an Easytrap Kit (Takara Biochemicals, Tokyo, Japan) and used as probe DNA to detect the integrated NAE6 into chromosomal DNA. To identify the virulence expression, the gene psvA (ORF3) was used as a hybridization probe. The labelling of probe DNA with horseradish peroxidase, hybridization and detection by exposure on autoradiography film were done using an ECL direct nucleic acid labelling and detection kit (Amersham, London, UK) (GE Healthcare Life Sciences, Little Chalfont, UK). Acknowledgments: The authors thank Hiroshi Kamiunten for his support and guidance in this research. Author Contributions: T.D.K. conducted the experiments. T.D.X. wrote and revised the manuscript. Both authors had checked and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflict of interests.
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