Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages

Virology 494 (2016) 56–66

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Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages Anjana Bhunchoth a,b,c, Romain Blanc-Mathieu d, Tomoko Mihara d, Yosuke Nishimura d, Ahmed Askora e, Namthip Phironrit a, Chalida Leksomboon f, Orawan Chatchawankanphanich a, Takeru Kawasaki g, Miyako Nakano g, Makoto Fujie g, Hiroyuki Ogata d,n, Takashi Yamada g,nn a

Plant Research Laboratory, National Center for Genetic Engineering and Biotechnology, NSTDA, Pathum Thani 12120, Thailand Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE), Bangkok 10900, Thailand d Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan e Department of Microbiology and Botany, Faculty of Science, Zagazig University, Zagazig 44519, Egypt f Department of Plant Pathology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand g Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima 739-8530, Japan b c

art ic l e i nf o

a b s t r a c t

Article history: Received 29 December 2015 Returned to author for revisions 28 March 2016 Accepted 31 March 2016

Jumbo phages infecting Ralstonia solanacearum were isolated in Thailand (ϕRSL2) and Japan (ϕRSF1). They were similar regarding virion morphology, genomic arrangement, and host range. Phylogenetic and proteomic tree analyses demonstrate that the ϕRSL2 and ϕRSF1 belong to a group of evolutionary related phages, including Pseudomonas phages ϕKZ, 201ϕ2-1 and all previously described ϕKZ-related phages. Despite conserved genomic co-linearity between the ϕRSL2 and ϕRSF1, they differ in protein separation patterns. A major difference was seen in the detection of virion-associated-RNA polymerase subunits. All β- and β0 -subunits were detected in ϕRSF1, but one β0 -subunit was undetected in ϕRSL2. Furthermore, ϕRSF1 infected host cells faster (latent period: 60 and 150 min for ϕRSF1 and ϕRSL2, respectively) and more efficiently than ϕRSL2. Therefore, the difference in virion-associated-RNA polymerase may affect infection efficiency. Finally, we show that ϕRSF1 is able to inhibit bacterial wilt progression in tomato plants. & 2016 Elsevier Inc. All rights reserved.

Keywords: Jumbo phages ϕKZ-like phages Ralstonia solanacearum Genomic analysis Virion-associated-RNA polymerase

1. Introduction Bacteriophages (phages) are the most abundant organisms in the biosphere and have crucial influences on bacterial evolution and ecology (Wommack and Colwell, 2000; Hendrix, 2002; Ashelford et al., 2003; Suttle, 2005). Tailed phages belonging to the order Caudovirales contain double-stranded DNA (dsDNA) and represent the most numerous, most widespread, and probably the oldest group of bacteriophages (Hendrix, 1999). There are three families (i.e., Myoviridae, Podoviridae, and Siphoviridae) in the order Caudovirales, and myoviruses comprise approximately 25% of this order (Ackermann, 2003; 2011). T4-like phages classically representing Myoviridae phages have n

Corresponding author. Correspondence to :Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter Hiroshima University, 1-3-1 Kagamiyama, HigashiHiroshima 739-8530, Japan. E-mail addresses: [email protected] (H. Ogata), [email protected] (T. Yamada). nn

http://dx.doi.org/10.1016/j.virol.2016.03.028 0042-6822/& 2016 Elsevier Inc. All rights reserved.

a contractile tail sheath and infect a broad range of bacterial hosts. Studies using T4-like phage genomes as models have suggested that myovirus genomes are mosaic of conserved core genes, which include structural genes for head and tail proteins and enzyme genes for DNA and nucleotide processing, and the remaining variable accessory noncore genes (Filee et al., 2006). The functions of non-core genes are largely unknown, although it is assumed that they provide a selective benefit to phages (Hendrix, 2009). Several myoviruses are known to have a large genome over 200 kbp, and are designated as “jumbo phages” (Hendrix, 2009). These include Pseudomonas aeruginosa phage ϕKZ (280 kbp, Mesyanzhinov et al., 2002) and EL (211 kbp, Hertveldt et al., 2005), Pseudomonas chlororaphis phage 201ϕ2-1 (317 kbp, Thomas et al., 2008), Pseudomonas fluorescens phage OBP (284 kbp, Cornelisssen et al., 2012), Klebsiella phage vB_KleM-RaK2 (346 kbp, Simoliunas et al., 2013), Vibrio parahaemolyticus phage KVP40 (386 kbp, Miller et al., 2003), Stenotrophomonas maltophilia phage ϕSMA5 (250 kbp, Chang et al., 2005), and Yersinia enterocolitica phage R137 (270 kbp, Kiljunen et al., 2005). Jumbo phages have also been

A. Bhunchoth et al. / Virology 494 (2016) 56–66

reported for plant-associated bacteria such as Sinorhizobium meliloti (phage N3, 207 kbp; Martin and Long, 1984), Erwinia amylovora (PhiEaH1, 218 kbp; Meczker et al., 2014 and vB_Eam_Ea35-70, 271 kbp; Yagubi et al., 2014), and Ralstonia solanacearum (ϕRSL1, 231 kbp; Yamada et al., 2010). The largest myovirus genome sequenced to date is that of Bacillus megaterium phage G (498 kbp; accession no. JN638751; Sun and Serwer, 1997). Some of these jumbo phages encode many proteins with considerable similarity to ϕKZ proteins, and are called ϕKZ-related viruses (Cornelissen et al., 2012; Jang et al., 2013). However, others have very unique features that suggest a high level of diversity among jumbo phages. For example, ϕRSL1 consists of an icosahedral head (diameter from vertex to vertex: 123 nm) and a contractile tail (length: 105 nm; diameter: 24.5 nm) (Effantin et al., 2013), and differs morphologically from the phages of the Phikzlikevirus genus (Krylov et al., 2007; Fokine et al., 2007). ϕRSL1 can efficiently and stably infect a diverse range of phytopathogenic R. solanacearum strains (Yamada et al., 2010; Fujiwara et al., 2011), and therefore, is considered a promising agent for the biocontrol of bacterial wilt. R. solanacearum is a soil-borne Gram-negative bacterium (belonging to Betaproteobacterium) that is the causative agent of bacterial wilt of many economically important crops (Yabuuchi et al., 1995; Hayward, 2000). The bacterium has an unusually wide host range and shows great phenotypic and genotypic diversity between strains (Hayward, 2000). With the aim of developing biocontrol methods effective against R. solanacearum, we identified various phages, including novel jumbo phages, in natural environments (Yamada, 2012; Bhunchoth et al., 2015). In this study, two jumbo phages, ϕRSL2 (isolated in Thailand) and ϕRSF1 (isolated in Japan), were characterized and compared with other jumbo phages.

2.2. General genomic features of

57

ϕRSL2 and ϕRSF1

The nucleotide sequences of the two genomes were determined using shotgun sequencing of DNA purified from phage particles. Sequences were assembled into a circular contig of 223,932 bp for ϕRSL2 (accession no. AP014693) and 222,888 bp for ϕRSF1 (accession no. AP014927). The ϕRSL2 and ϕRSF1 genomes had G þC contents of 52.06% and 52.26%, respectively, which were significantly lower than that of the host genome (e.g., 66.97% for strain GMI1000; accession no. NC_003295). The sequences of the two phage genomes were very similar and exhibited nearly complete co-linearity (Fig. S3). In total, 237 potential open reading frames (ORFs) were detected in each of the ϕRSL2 and ϕRSF1 genomes (Fig. S4). No tRNA or tRNA-related sequences were detected in either genome. As expected from the high level of genome sequence similarity (Fig. S3), ϕRSL2 and ϕRSF1 showed a similar gene order globally and locally, although several genes were specific to one of the genomes (Table S2). Twenty-three percent (54/237) of ϕRSL2 ORFs and 24% (57/237) of ϕRSF1 ORFs were located in a clockwise direction, with the remaining ORFs encoded in a counterclockwise direction. Based on similarities to biologically characterized proteins in available databases, only 71 and 67 ORFs of ϕRSL2 and ϕRSF1, respectively, were functionally annotated (Tables S2). BLASTP searches against phage and viral genomes in the NCBI/RefSeq database identified many ϕRSL2 and ϕRSF1 ORFs significantly similar to Phikzlikevirus ORFs. For instance, 37 ORFs of ϕRSL2 showed their best hit to ORFs in ϕKZ, 201ϕ2-1 or ϕPA3. We also identified putative promoter sequences. The sequences are represented in Fig. S5 as sequence logos with a comparison to a similar motif found in intermediate genes of ϕKZ (Mesyanzhinov et al., 2002; Ceyssens et al., 2014). The motifs detected for 16 ϕRSL2 ORFs and 20 ϕRSF1 ORFs were very similar to each other and were related to that of ϕKZ.

2. Results 2.1. Isolation and initial characterization of

ϕRSL2 and ϕRSF1

ϕRSL2 is a jumbo myovirus that was isolated in Chiang Mai, Thailand (Bhunchoth et al., 2015). It formed small clear plaques with a wide range of host strains (including those isolated in Japan) in assays using low concentrations ( o0.45%) of top agar. Its morphology and genome organization were very different from those of ϕRSL1, which is a jumbo phage previously isolated and characterized in Japan (Yamada et al., 2010). Therefore, we were interested in isolating new phages related to ϕRSL2 in Japan for comparative studies. Using a large-phage screening method, ϕRSF1 was detected from soil samples collected in Sera, Japan. ϕRSF1 formed very small plaques (o0.1 mm) with host strains using 0.45% top agar plates, but formed larger plaques (1–2 mm) when the top agar concentration was decreased to 0.3%. ϕRSF1 formed plaques with 18 of 21 R. solanacearum strains isolated in Japan (Table S1), thus exhibiting a wider host range than ϕRSL2 (13 of 21). The jumbo phage nature of ϕRSF1 was confirmed by its large genome size and morphology. In contour-clamped homogeneous electric field (CHEF) gel electrophoresis analyses, the ϕRSF1 genomic DNA produced a single band of approximately 220 kbp, which was almost the same size as that of ϕRSL2 DNA, but slightly smaller than the ϕRSL1 DNA band (approximately 240 kbp) (Fig. S1). An analysis of ϕRSF1 particle morphology using electron microscopy revealed characteristics of a myovirus, with an icosahedral head (diameter: approximately 115 75 nm, n¼ 10) and a long contractile tail (length: 180 710 nm, n¼ 10; width: 25 72 nm, n ¼10) (Fig. S2). The ϕRSF1 particles were very similar to those of ϕRSL2 (Bhunchoth et al., 2015), with minor exceptions such as in the baseplate structures. Thus, ϕRSF1 resembled ϕRSL2 in terms of morphology and genome size.

2.3. Evolutionary relationships between related phages

ϕRSL2/ϕRSF1 and ϕKZ-

To examine the evolutionary relationship between ϕRSL2/ ϕRSF1 and ϕKZ-related phages, we first performed PSI-BLAST searches from each of the ORFs in 201ϕ2-1, the largest phage of the Phikzlikevirus genus, against all currently sequenced Caudovirales genomes. This search revealed 81 ϕRSL2 and 77 ϕRSF1 ORFs showing significant sequence similarities to 201ϕ2-1 ORFs (Table S3). These numbers are greater than those for phage OBP (67 ORFs) and EL (69 ORFs), which were previously described as ϕKZ-related phages (Cornelissen et al., 2012). Dot-plot analyses based on TBLASTX confirmed the presence of long stretches of colinear genomic segments between ϕRSL2/ϕRSF1 and other ϕKZrelated phages (Fig. S6). It is notable that Bacillus phage G, which is not a ϕKZ-related phage, exhibits no such conserved segments, albeit it encodes 32 ORFs homologous to 201ϕ2-1 ORFs (Table S3). By focusing on the genomes ϕRSL2, ϕRSF1 and five ϕKZ-related phages, we identified 52 groups of orthologs conserved in all of these phages (Table S4). These conserved genes include those for terminases, major capsid proteins, many virion structural proteins, RNase H1, SbcC as well as RNA polymerase (RNAP) β and β0 subunits. Conserved physical linkages of these orthologs support again the putative evolutionary relationship between ϕRSL2/ ϕRSF1 and ϕKZ-related phages (Fig. 1). Homologous genomic fragments identified in this way typically contained a cluster of several genes. For example, a fragment encoding ϕRSL2 ORF23ORF28 was similar to a ϕKZ fragment containing ORF25–ORF30, and a ϕRSL2 fragment containing ORF34–ORF39 was homologous to a ϕKZ fragment encoding ORF176–ORF181. Finally, we performed a proteomic tree and phylogenetic tree analyses. A phage proteomic tree based on genome-wide sequence

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Fig. 1. Genome sequence comparison among seven phiKZ-related viral genomes exhibiting co-linearity detected by TBLASTX. Phage abbreviations are as follows: 201ϕ2-1, Pseudomonas phage 201ϕ2-1 (Phikzlikevirus); ϕPA3, Pseudomonas phage ϕPA3 (Phikzlikevirus); ϕKZ, Pseudomonas phage ϕKZ (Phikzlikevirus); RSL2, Ralstonia phage ϕRSL2; RSF1, Ralstonia phage ϕRSF1; EL, Pseudomonas phage EL; OBP, Pseudomonas phage OBP. ϕRSL2 and ϕRSF1 are circularly permuted at 30 kb and reverse stranded for the clarity. 201ϕ2-1, ϕPA3, ϕKZ, EL and OBP were previously described to as members of ϕKZ-related phages. Color bar shows %-identity of TBLASTX. Homologous genes among the seven genomes detected by PSI-BLAST are colored by functional annotations as represented in the figure. An asterisk represents three RNAP ORFs of ϕPA3 detected by MetaGeneMark (these three ORFs are one concatenated gene in RefSeq). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

similarity scores revealed a clear clade containing 13 phages showing conserved genomic co-linearity (Figs. 2A and S6). These are ϕRSL2, ϕRSF1, ϕKZ-related phages and other phages, for which evolutionary relationships to ϕKZ-related phages were previously suggested (Cornelissen et al., 2012; Jang et al., 2013). Among the 13 phages, OBP and EL were found as a most deeply branching group in the proteomic tree, while ϕRSL2 and ϕRSF1 were placed more closely to three phages of Phikzlikevirus (i.e., ϕKZ, 201ϕ2-1 and ϕPA3). We also performed maximum likelihood phylogenetic tree reconstructions for phage genes (tail sheath proteins, Fig. 2B; terminases, Fig. 2C; conserved structural proteins, Fig. S7D; major capsid proteins, Fig. S7E) as well as genes showing homologs in bacteria (ribonucleotide reductase β and α subunits, Fig. S7A and S7B; DNA ligases, Fig. S7C). Again, compared to OBP and EL, ϕRSL2 and ϕRSF1 were closer to phages of the Phikzlikevirus genus. Overall, these sequence similarity relationships, proteomic tree and gene phylogenies suggest that ϕRSL2 and ϕRSF1 are new members of the group of ϕKZ-related phages. Phages with a genome larger than 200 kb were scattered across the proteomic tree (Fig. 2A). For instance, we did not detect any close relationships among ϕKZ-related phages, Bacillus phage G (498 kb) and Cronobacter phage GAP32 (359 kb) either in the proteomic tree or in the phylogenetic trees. This demonstrates that jambo phages do not form a monophyletic group and that they evolved independently several times in different lineages of phages. 2.4.

ϕRSL2 and ϕRSF1 gene annotations

Here we describe notable genes found in the genomes of ϕRSL2 and ϕRSF1.

2.4.1. RNA polymerase subunits Two distinct and virally encoded multisubunit RNAPs were proposed to function during the infection cycle of ϕKZ: a phagepacked-RNAP responsible for early gene expression in the absence of host RNAP activity and another early-expressed RNAP for middle and late phases of phage gene expression (Ceyssens et al., 2014). All genes for these subunits (virion-associated RNAP, Gp178, Gp149, Gp180, and Gp80 as well as early-expressed-RNAP, Gp123, Gp71-Gp73, Gp55, and Gp74) were present in both ϕRSL2 and ϕRSF1. The possible gene-for-gene correspondence among these phages is shown in Table 1. As described below, all proteins corresponding to ϕKZ virion-associated RNAP subunits were actually detected in the ϕRSF1 virion. 2.4.2. Proteins involved in DNA replication, recombination, and repair None of the ORFs in the ϕRSL2 and ϕRSF1 genomes showed significant sequence similarities to known DNA polymerases. ϕRSL2 and ϕRSF1 predicted proteins involved in DNA replication included RNase H (ϕRSL2-ORF54 and ϕRSF1-ORF57), SbcC-ATPase (ϕRSL2-ORF61 and ϕRSF1-ORF63), DNA ligase (ϕRSL2-ORF95 and ϕRSF1-ORF105), and DnaB helicase (ϕRSL2-ORF119 and ϕRSF1ORF126). ϕRSF1-ORF68 was similar to GIY-YIG type nucleases, which are involved in many biological processes, including DNA repair and recombination, transfer of mobile genetic elements, and digestion of foreign DNA. 2.4.3. Nucleotide metabolism and DNA modification enzymes ϕRSL2 and ϕRSF1 homologs of at least six characterized enzymes for deoxyribonucleotide triphosphate conversion and pyrimidine synthesis were detected in the ϕRSL2 and ϕRSF1

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Fig. 2. Proteomic and phylogenetic relationships between ϕRSL2/ϕRSF1 and other phages. (A) A proteomic tree produced by the BIONJ program (Gascuel, 1997) based on TBLASTX genomic sequence comparisons of 59 large bacteriophage genomes. Phages with genome larger than 200 kb have their name colored with red. Branch lengths from the root were scaled logarithmically. In this logarithmic representation, nodes that were at distances smaller than 0.001 from the root were agglomerated into the root point. (B and C) Maximum likelihood phylogenetic trees of the tail sheath and terminase large subunit proteins respectively. Statistical support at node is given as bootstrap values. Number at scale bar indicate the number of substitutions per site. Trees are midpoint rooted. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

genomes, including a thymidylate kinase (ϕRSL2-ORF182 and ϕRSF1-ORF190), a thymidylate synthase (ϕRSL2-ORF153 and ϕRSF1-ORF164), dihydrofolate reductases (ϕRSL2-ORF73,-ORF74

and ϕRSF1-ORF77,-ORF78), a ribonucleotide reductase α subunit (ϕRSL2-ORF111 and ϕRSF1-ORF118) and a β subunit (ϕRSL2ORF110 and ϕRSF1-ORF117), and an anaerobic ribonucleoside

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Table 1 β and β0 RNAP-like subunits. Viruses

Virion-associated-RNAP β0 subunit (RpoC)

β subunit (RpoB) N-region ϕKZ ϕRSL2 ϕRSF1 a b

ORF178 ORF37 ORF40

Early-expressed RNAP

C-region ORF149 ORF48 ORF51

N-region ORF180 ORF38 ORF41

β0 subunit (RpoC)

β subunit (RpoB) C-region ORF80 ORF192 ORF199

N-region a

ORF123 ORF115a ORF122a

C-region ORF71-73 ORF209 ORF215

b

N-region

C-region

ORF55 ORF221 ORF227

ORF74 ORF208 ORF214

We did not identify sequence similarity to known RNA polymerases, albeit ϕKZ ORF123 has been previously annotated an RpoB fragment (Ceyssens et al., 2014). Split into two ORFs.

diphosphate reductase subunit H (ϕRSL2-ORF112 and ϕRSF1ORF119). There were no homologs of enzymes involved in DNA modification, such as adenine and cytosine methylation or cytosine hydroxymethylation, in either genome. The presence of ORFs related to DNA-methyltransferases could not be confirmed. 2.4.4. Lysis and host–phage interaction ϕRSL2-ORF39 ( 2039 amino acids) and ϕRSF1-ORF42 (1930 amino acids) showed similarities to soluble lytic murein transglycosylases (chitinase-like glycosylases or LysM/invasin domains) and were found to be homologous to the putative cell-puncturing protein Gp181 (2237 amino acids) of ϕKZ (Fokine et al., 2007). These large proteins were found associated with ϕRSL2 and ϕRSF1 virions. Proteins encoded by ϕRSL2-ORF52 and ϕRSF1-ORF55 were similar to the lytic transglycosylase-like SLT domains. This type of soluble transglycosylase degrades murein via cleavage of

the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine, with the concomitant formation of a 1,6anhydro bond in the muramic acid residue. 2.4.5. Structural proteins A comparative analysis of the ϕRSL2 and ϕRSF1 genome sequences enabled the initial annotation of 46 and 43 virionassociated protein genes, respectively (Table S2). These include genes for structural proteins such as major capsid protein (ϕRSL2ORF117 and ϕRSF1-ORF124), cell puncturing device (ϕRSL2-ORF39 and ϕRSF1-ORF42), tail tube (ϕRSL2-ORF28 and ϕRSF1-ORF31), tail fiber (ϕRSL2-ORF158 and ϕRSF1-ORF169), tail sheath (ϕRSL2ORF27 and ϕRSF1-ORF30), baseplate wedge (ϕRSL2-ORF59 and ϕRSF1-ORF89), baseplate assembly protein (ϕRSL2-ORF62 and ϕRSF1-ORF64) and other possible structural proteins. Reversedphase nano-liquid chromatography directly coupled with liquid

Fig. 3. Proteomic analysis of virion proteins of ϕRSL2 (A) and ϕRSF1 (B). Virion proteins separated by SDS-PAGE were visualized with Coomassie Brilliant Blue. The protein bands were excised from the gel, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry (LTQ Orbitrap XL). Assignment of tandem mass spectrometry data to tryptic peptides encoded by phage open reading frames was completed using an established procedure (Ahmad et al., 2014). Asterisks indicate the β and β0 subunits of virion-associated-RNAP. VAP: virion associated protein. VSP: virion structural protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 2 Mass spectrometry data for RSL2. RSL2 protein

Predicted function

Theo. mol. mass (Da)

Obs. mol. mass (kDa)

No. of unique peptides

Sequence coverage (%)

ORF12 ORF21 ORF24 ORF26 ORF27 ORF28 ORF33 ORF34 ORF35 ORF37 ORF39 ORF40 ORF41 ORF48 ORF57 ORF58 ORF62 ORF63 ORF84 ORF90 ORF109 ORF117 ORF148 ORF158 ORF159 ORF166 ORF169 ORF170 ORF172 ORF176 ORF178 ORF180 ORF185 ORF191 ORF192 ORF224

Virion associated protein Virion associated protein Virion structural protein Virion structural protein Tail sheath protein Tail tube protein Virion structural protein Virion associated protein Virion associated protein Putative RNA polymerase beta subunit Cell-puncturing device Virion associated protein Virion associated protein Putative RNA polymerase beta subunit Virion structural protein Virion associated protein Putative phage baseplate assembly protein Virion structural protein Phage-encoded peptidoglycan binding protein Virion structural protein Putative N-acetyltransferase YedL Major capsid protein Virion associated protein Tail fiber protein Virion associated protein Virion associated protein Virion structural protein Virion associated protein Virion structural protein Virion associated protein Virion structural protein Putative SPRY domain-containing protein Virion structural protein Virion associated protein Putative RNA polymerase beta0 subunit Virion associated protein

31,020 47,865 61,138 33,837 78,203 31,756 28,894 26,938 52,474 164,013 221,140 69,069 42,597 25,631 47,550 22,033 31,705 48,028 28,655 31,326 25,429 82,440 21,372 119,882 39,732 59,693 47,946 47,202 34,028 66,525 50,655 21,488 36,136 34,580 48,722 41,719

33 46 70 38 80 38 28 33 60 240 240 70 43 31 45 28 43 48 31 38 33 70 31 140 46 60 41 39 38 45 46 28 43 38 52 43

2 8 3 13 72 13 4 4 45 69 13 3 5 4 11 4 1 10 1 11 1 48 3 4 5 21 4 54 30 55 17 1 1 4 16 4

5.6 16.1 5.3 32.8 74.3 32.6 11.9 13.3 52.4 34.6 5.4 4.9 10.0 15.4 25.4 15.2 3.7 20.2 4.6 33.5 3.8 57.3 18.2 3.9 11.1 39.8 8.2 61.6 55.7 49.3 21.7 5.6 4.1 13.2 35.0 10.9

The proteins detected by MS are listed with their theoretical molecular mass and observed molecular mass. The number of identified peptides in each protein and the corresponding protein sequence coverage are indicated.

chromatography-tandem mass spectrometry analysis of the proteins of ϕRSL2 and ϕRSF1 virions separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) resulted in the identification of 36 ϕRSL2 virion proteins (Fig. 3A) and 41 ϕRSF1 virion proteins (Fig. 3B), respectively. These included 60% (28/46) of the ϕRSL2 ORFs and 77% (33/43) of the ϕRSF1 ORFs that were predicted to be virion-associated based on the homology searches described above (Table S2) and additional proteins showing marginal homology to some enzymes and other unknown proteins (finally annotated as virion-associated proteins) (Tables 2 and 3). In the case of ϕRSL2 virion, the β-subunit (ORF37 and ORF48) and β0 -subunit (ORF192 but not ORF38) of virionassociated-RNAP-type proteins (Ceyssens et al., 2014) were detected among the ϕRSL2 structural proteins (Fig. 3A). In contrast, all β-subunits (ORF40 and ORF51) and β0 -subunits (ORF41 and ORF199) of virion-associated- RNAP-type proteins were detected in ϕRSF1 (Fig. 3B, Table 3). The SDS-PAGE protein separation patterns for virion proteins were considerably different between ϕRSL2 and ϕRSF1 (Figs. 3A and B). This was mainly due to size differences between corresponding proteins in ϕRSL2 and ϕRSF1, even though the amino acid sequences were highly conserved. To detect ϕRSL2-ORF38 protein, gels run with higher amounts of ϕRSL2 proteins were sliced in pieces around 40– 100 kDa regions and subjected to MS analysis. In three trials, ϕRSL2-ORF38 protein could not be detected. 2.4.6. Paralogous gene families The ϕRSL2 and ϕRSF1 genomes did not encode large paralogous families (Table S5). For ϕRSL2, there were 15 groups of

paralogs. The largest groups of paralogs with three members each were putative SPRY domain-containing proteins (ORF001, 180, 181), virion structural proteins (ORF183, 184, 185) and hypothetical proteins (ORF202, 203, 204). For ϕRSF1, there were 12 groups of paralogs. The largest group of paralogs with three members was again putative SPRY domain-containing proteins (ORF001, 190, 191). 2.4.7. Other genes Several ORFs encoding proteins homologous to known functional proteins were detected in ϕRSL2 and ϕRSF1, including an Nacetyltransferase (ϕRSL2-ORF20 and ϕRSF1-ORF21), the ‘cupin’ superfamily protein (ϕRSL2-ORF66 and ϕRSF1-0RF69), an Fe–S oxidoreductase (ϕRSL2-ORF67 and ϕRSF1-ORF70, ORF79), a radical SAM superfamily protein (ϕRSL2-ORF75, ORF77 and ϕRSF1ORF75, ORF81), a 2OG–Fe(II) oxygenase (ϕRSL2-ORF76, ORF78 and ϕRSF1-ORF80, ORF82), a concanavalin A-like protein (ϕRSL2ORF83 and RSF1-ORF89, ORF90), and a haloacid reductase-like hydrolase (ϕRSL2-ORF149 and ϕRSF1-ORF160) (Table S2). The actual expression patterns and functions of these gene products during the phage infection cycle are unknown. 2.5.

ϕRSL2 and ϕRSF1 infection cycles

The ϕRSL2 and ϕRSF1 infection cycles were compared using single-step growth experiments with R. solanacearum strain MAFF 730138 as the host. As shown in Fig. 4, ϕRSF1 infection proceeded significantly faster and more efficiently than ϕRSL2. In the former

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Table 3 Mass spectrometry data for RSF1. RSF1 protein

Predicted function

Theo. mol. mass (Da)

Obs. mol. mass (kDa)

No. of unique peptides

Sequence coverage (%)

ORF22 ORF27 ORF28 ORF29 ORF30 ORF31 ORF36 ORF37 ORF40 ORF41 ORF42 ORF43 ORF44 ORF51 ORF54 ORF59 ORF64 ORF65 ORF66 ORF67 ORF94 ORF100 ORF101 ORF124 ORF125 ORF159 ORF169 ORF170 ORF176 ORF177 ORF180 ORF182 ORF183 ORF184 ORF185 ORF186 ORF188 ORF198 ORF199 ORF229 ORF230

Virion associated protein Virion structural protein Virion structural protein Virion structural protein Tail sheath protein Tail tube protein Virion structural protein Virion associated protein Putative RNA polymerase beta subunit Putative RNA polymerase beta0 subunit Cell-puncturing device Virion associated protein Virion associated protein Putative RNA polymerase beta subunit Virion associated protein Virion structural protein Putative phage baseplate assembly protein Virion structural protein Virion structural protein Virion structural protein Virion associated protein Virion structural protein Virion structural protein Major capsid protein Virion associated protein Virion associated protein Tail fiber protein Virion associated protein Virion structural protein Virion associated protein Virion associated protein Virion structural protein Virion structural protein Virion associated protein Virion structural protein Virion associated protein Virion structural protein Virion associated protein Putative RNA polymerase beta0 subunit Virion structural protein Virion associated protein

47,784 63,277 94,294 34,056 77,967 31,708 29,058 26,957 165,279 60,803 229,858 70,511 39,231 24,436 35,752 47,809 36,041 47,795 73,575 40,390 32,876 81,524 111,961 82,301 24,949 23,563 120,176 39,762 51,541 59,200 46,589 34,196 40,022 27,315 108,150 64,996 50,468 34,812 49,906 76,959 40,370

44 67 110 35 90 37 29 32 230 67 260 67 40 32 34 48 40 47 90 47 42 95 90 67 29 29 130 44 55 60 37 37 29 47 130 48 48 35 60 67 40

14 24 26 13 81 24 5 8 39 24 111 18 14 7 18 11 4 11 23 9 12 6 9 60 4 6 7 11 5 5 70 28 10 2 12 25 21 1 11 7 8

27.7 41.8 27.7 32.4 83.0 72.6 17.7 32.5 22.0 29.4 49.4 31.2 26.5 25.6 49.4 25.6 15.4 26.4 49.7 27.9 24.5 8.7 13.4 56.5 14.6 23.5 7.6 24.9 11.8 10.5 66.0 70.6 26.7 10.8 12.7 30.6 33.9 4.0 20.3 15.1 15.8

The proteins detected by MS are listed with their theoretical molecular mass and observed molecular mass. The number of identified peptides in each protein and the corresponding protein sequence coverage are indicated.

case, one infection cycle took 240 min with a latent period of 90min, whereas the latter consisted of a 150-min latent period and a 270-min infection cycle. The burst size was approximately 75 plaque-forming units (pfu)/cell for ϕRSF1 and 40–50 pfu/cell for ϕRSL2. 2.6. Prevention of bacterial wilt of tomato plants by treatment

ϕRSF1

Lytic phages with a wide host range may be used directly to prevent bacterial wilt caused by R. solanacearum in the soil. To explore this possibility, we used ϕRSF1 to treat tomato plants challenged with the highly virulent R. solanacearum strain MAFF 211514. As shown in Fig. 5, ϕRSF1 treatment effectively prevented wilting of infected tomato plants. Plants that did not receive phage treatment started to show wilting symptoms at 4 days post inoculation (dpi) and most plants exhibited severe wilting symptoms at 10 dpi (Fig. 5A). Almost identical results were observed with plants that were infected with R. solanacearum before phage treatment (data not shown). In contrast, most of the ϕRSF1treated plants showed no signs of wilting throughout the experimental period (up to 30 dpi) (Fig. 5B). Essentially the same results were reproducibly obtained in three independent experiments using ϕRSF1 (data not shown).

3. Discussion 3.1.

ϕRSL2 and ϕRSF1 as ϕKZ-related phages

Several jumbo phages are considered to be ϕKZ-related phages according to their common conserved features (Cornelissen et al., 2012). Typically, ϕKZ-related phages have a very large icosahedral head (diameter: 120–125 nm) and a long (4 190 nm) contractile tail surrounded by fibers (Krylov et al., 2007). The genomes of these phages are large (4200 kbp), circularly permuted, and terminally redundant linear double-stranded DNA. Their G þC content is always lower (36–48%) than that of the host (60–88%). The ϕKZ-related phages are further subdivided (based on genomic and genetic similarity) into ϕKZ-like viruses, including ϕKZ, 201ϕ2-1, ϕPA3, and EL-like viruses such as EL and OBP (Lavigne et al., 2009; Cornelissen et al., 2012). The ϕKZ-related phage features are conserved in ϕRSL2 and ϕRSF1. However, the G þC content is higher in ϕRSL2 (52.06%) and ϕRSF1 (52.26%) than in previously analyzed ϕKZ-related phages. As shown in Figs. 1 and 2, S6 and S7, phylogenetic and comparative analyses at both genomic and gene levels revealed ϕRSL2 and ϕRSF1 are closely related to previously recognized ϕKZ-related phages. ϕRSL2 and ϕRSF1 contain many genes conserved in ϕKZ-like viruses, including those involved in nucleotide metabolism and the β and β0 subunits of the

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conservations only within the same group, with considerable genomic differences between groups (Jeanniard et al., 2013). It is noteworthy that the range of hosts isolated in Japan is wider for ϕRSF1 (isolated in Japan) than for ϕRSL2 (isolated in Thailand), indicating an evolutionary adaptation of these phages to geographical variants of R. solanacearum (Table S1). 3.2.

Fig. 4. Single-step growth curves of ϕRSL2 and ϕRSF1 with Ralstonia solanacearum strain MAFF 730138 as the host. Phage (grown with the same host) was added at a multiplicity of infection of 0.1 and allowed to adsorb for 10 min at 28 °C. Samples were collected at specific intervals and the titers were determined using doublelayered agar plates.

multisubunit RNAP. From our proteomic and phylogenetic tree analyses, ϕEaH1 and ϕEaH2 were found to be evolutionarily related to ϕKZ-related phages. It should be noted that ϕEaH1 and ϕEaH2 belong to the Siphoviridae family. Whether these two siphoviruses should be classified in the ϕKZ-related phages might be controversial, given that the previously described ϕKZ-related phages are myoviruses. This also suggests a problem in the current phage classification based on morphology, which may not necessarily reflect evolutionary relationships of phages. ϕRSL2 and ϕRSF1 were isolated from different countries, but their genome organizations were very similar, with a long range co-linearity maintained over almost the entire genome (Fig. 1A and S3). More fragmented but still highly conserved co-linear segments were also observed among ϕKZ-related phages and ϕRSL2/ϕRSF1 (Fig. 1). This situation is very similar to that of giant viruses of Chlorella spp. Each member of three groups of chloroviruses (i.e., NC-64A-viruses, Pbi-viruses, and SAG-viruses, distinguished by the host Chlorella species) shows a high level of genomic

A single-step growth curve indicated a faster (by 60 min) and more efficient (1.5-fold larger burst size) infection by ϕRSF1 than ϕRSL2 in the same host. The shorter latent period in ϕRSF1 infection (Fig. 4) suggests earlier expression of early genes in this phage compared with ϕRSL2. Host-independent early gene expression mediated by virion-associated-RNAP was proposed by Ceyssens et al. (2014) in ϕKZ infection. Both ϕRSF1 and ϕRSL2 are, as described above, ϕKZ-related phages and encode β and β0 subunits of RNAPs (Table 1). However, it is noteworthy that a portion of the β0 subunit (ORF38) was not detected in ϕRSL2 virion, but ϕRSF1 virion possessed a full set of β and β0 subunits (Fig. 2). Some changes might have occurred in the ϕRSL2 protein in quantity and/or quality (by processing or modification) to be out of detection. This change in virion-associated-RNAP of ϕRSL2 may cause dependency of early gene expression at least partially on host RNAP, resulting in a more time-consuming process. To clarify the possible differences in the activity of phage RNAP between ϕRSF1 and ϕRSL2 during infections, we assessed the effects of rifampicin on phage replication. The minimal inhibitory concentration of rifampicin on strain MAFF 730138 growth was 3 mg/ ml. At this concentration, neither ϕRSF1 nor ϕRSL2 could replicate (data not shown). Therefore, we could not confirm the involvement of virion-associated- RNAP in early gene expression during phage infection. Additionally, this virion-associated- RNAP strategy raises the question of how RNAP proteins are associated with the genomic DNA to be able to be packaged in the virion. Furthermore, during the initial stage of infection, the DNA-protein complex should be introduced into the host cytoplasm through the host cell membrane. Some specific interactions between RNAP and genomic DNA may prevent extensive genomic rearrangements in the phages. Because both phages showed almost the same degree of inhibitory effects on host bacterial virulence in plants, the selective advantage of the RNAP difference in natural ecosystems is currently unknown.

B

5 Disease index

4

5 3

4 3

2

2 1

1

Number of tomato plant

Number of tomato plant

A

ϕRSL2 and ϕRSF1 infection cycles

5 Disease index

4

5 3

4 3

2

2 1

1

0

0 0

0 0

4

8 12 16 20 24 28 Days post infection

0

4

8 12 16 20 24 28 Days post infection

Fig. 5. Prevention of bacterial wilt by ϕRSF1 treatment. Four-week-old tomato plants (n¼ 5) pretreated with tap water (control) (A) or ϕRSF1 (B) were inoculated with Ralstonia solanacearum strain MAFF 211514. Wilting symptoms were graded from 0 to 5 as follows: 1, only one petiole was wilting; 2, two or three petioles were wilting; 3, all but two or three petioles were wilting; 4, all petioles were wilting; and 5, the plant died.

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3.3. Comparison of

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ϕRSL2 and ϕRSF1 with ϕRSL1

In a previous study, we characterized ϕRSL1, which is another jumbo phage that infects R. solanacearum (Yamada et al., 2010). In terms of its morphology and genome, ϕRSL1 is significantly different from ϕRSL2 and ϕRSF1. It is a myovirus with an icosahedral head (diameter: 123 nm) and a relatively short contractile tail (length: 105 nm). The tail structure contains a neck/collar region between the head and tail from which fibers protrude. The fibers are packed in an orderly manner along the helical tail and are of an appropriate length that enables to contact with the host surface when the tail is contracted (Effantin et al., 2013). The ϕRSL1 genome is approximately 240 kbp, and contains 343 ORFs, most of which are orphans (without any homology to ϕΚΖ genes) (Yamada et al., 2010). ϕRSL1 exhibits stable growth-limiting effects on many R. solanacearum strains in laboratory cultures as well as prolonged disease prevention effects in plants (Fujiwara et al., 2011). As indicated in Fig. 4, ϕRSF1 also limited disease progression in tomato plants challenged by pathogenic bacteria. ϕRSL1 and ϕRSF1 will contribute to the effective biocontrol of bacterial wilt, which is one of the most serious crop diseases spreading worldwide.

4. Materials and methods 4.1. Bacterial strains, bacteriophages, and culture conditions The R. solanacearum strains used in this study, along with their hosts and taxonomic features, are listed in Table S1. The bacterial cells were cultured in CPG medium containing 0.1% (w/v) casamino acids, 1.0% (w/v) peptone, and 0.5% (w/v) glucose (Horita and Tsuchiya, 2002) at 28 °C with shaking at 200–300 rpm. For longterm storage, bacterial cultures were kept in sterile 20% (v/v) glycerol at  80 °C. ϕRSL2 was the previously described J6 phage (Bhunchoth et al., 2015), while ϕRSF1 was newly isolated from tomato fields in Sera, Hiroshima, Japan using an established isolation procedure (Bhunchoth et al., 2015). Briefly, 1 g soil was suspended in 2 ml distilled water and vigorously shaken for 1 h at room temperature to release bacteriophages. The mixture was filtered through a membrane filter (0.45-mm pore; Steradisc, Kurabo Co. Ltd., Osaka, Japan). The suspension (100 ml aliquots) was used in a plaque-forming assay, with R. solanacearum MAFF 106603 as the host, on CPG plates containing 1.5% agar overlaid with 0.45% CPG soft agar. Each phage was routinely propagated using R. solanacearum strain MAFF 106603 as the host. When the cultures reached an OD600 of 0.5, bacteriophages were added at a multiplicity of infection of 0.01–0.1. After culturing for a further 12–24 h, the cells were removed by centrifugation at 8000g for 15 min at 4 °C. The supernatant was filtered (0.45 mm membrane), and the pellet was dissolved in SM buffer (50 mM Tris–HCl at pH 7.5, 100 mM NaCl, 10 mM MgSO4, and 0.01% gelatin). For further purification, the phage suspension was layered on a 20–60% sucrose gradient and centrifuged at 40,000g for 1 h. The purified phages were stored at 4 °C. The phage particles were stained with Na-phosphotungstate or uranyl acetate and analyzed using a JEOL JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan) according to the method of Dykstra (1993). We used λ phage particles as an internal standard marker for size determination. 4.2. Single-step growth experiments Single-step growth experiments were performed as previously described (Yamada et al., 2010), with some modifications. Strain MAFF 730138 was used as the host. Bacterial cells (0.1 U of OD600) were harvested by centrifugation at 8000g for 15 min at 4 °C and

resuspended in fresh CPG medium (approximately 1  108 colonyforming units/ml) in a final volume of 10 ml. The phage was added at a multiplicity of infection of 0.1 and allowed to adsorb for 10 min at 28 °C. After centrifugation at 8000g for 15 min at 28 °C, samples were resuspended using the initial volume of CPG, and serial dilutions were prepared in a final volume of 10 ml. The cells were incubated at 28 °C. Samples were removed at 30-min intervals up to 5 h and the titers were determined using double-layered agar plates. 4.3. Isolation and characterization of genomic DNA from phage particles Standard techniques for DNA isolation, digestion with restriction enzymes, construction of recombinant DNA, and sequencing were completed according to Sambrook and Russell (2001). Genomic DNA was isolated from the purified phage particles by phenol extraction. To determine genome size, the purified phage particles were embedded in 0.5% low-melting-point agarose (InCert agarose, FMC Corp., Philadelphia, PA, USA). Following treatment with proteinase K (1 mg/ml; Merck Ltd., Tokyo, Japan) and 1% (w/v) sarkosyl, the nucleic acids were subjected to pulsedfield gel electrophoresis using the CHEF Mapper electrophoresis apparatus (Bio-Rad, Hercules, CA, USA) as described by Higashiyama and Yamada (1991). Shotgun sequencing of phage genomic DNA was completed at Hokkaido System Science Co., Ltd. (Sapporo, Japan) using a Roche GS Junior System. Draft sequences were assembled using GS De Novo Assembler, version 2.6. The analyzed sequences corresponded to 236 and 98 times the final contig sizes of ϕRSL2 (223,932 bp) and ϕRSF1 (222,888 bp), respectively. 4.4. Bioinformatics Potential ORFs longer than 90 bp (30 codons; ATG, GTG, TTG and CTG starts) were identified using Glimmer, version 3.02 (Delcher et al., 1999). Homology searches were performed using BLASTP/RPS-BLAST (Altschul et al., 1997) tools against NCBI/Cdd sequence domain database (version 3.14, Wheeler et al., 2007), UniProt sequence database (UniProt Consortium, 2014), and NCBI RefSeq complete viral genome database (Release 74). Additional homology searches were performed using PSI-BLAST started with 201ϕ2-1 amino acid sequences against NCBI RefSeq complete viral genome database for five iterations. To detected potential gene family expansion in ϕRSL2 and ϕRSF1, PSI-BLAST searches started from ϕRSL2 and ϕRSF1 amino acid sequences were performed with the same setting as above. An E-value lower than 1e-5 was used as the cutoff for notable similarity. tRNA genes were identified using tRNAScan-SE 1.4 (option:-B for bacterial tRNAs) (Lowe and Eddy, 1997). Circular genome maps were generated using CGView (Stothard and Wishart, 2005) and dot-plots by MUMmer 3.23 (Kurtz et al., 2004) as well as by an in-house script. Phylogenies (alignment and gene tree reconstruction) were conducted using the function build in the ETE3 toolkit package v3.0.0b33 (Huerta-Cepas et al., 2016): Sequences were aligned using MAFFT v6.861b (Katoh and Toh, 2008) with default parameters. Alignments were trimmed using trimAl v1.4.rev6 (Capella-Gutiérrez et al., 2009) if it improved the trees. Model of protein evolution were selected using ProtTest (Abascal et al., 2005) as implemented in pmodeltest v1.4. Selected models were WAG: the conserved virion structural protein and major capsid protein, VT: tail sheath and DNA ligase, LG: Terminase large subunit, ribonucleotide reductase β subunit and ribonucleotide reductase α subunit. Tree reconstruction was performed using RaxML v8.1.20 (Stamatakis, 2014) with the selected model and PROTGAMMA parameter. Branch supports were drawn from 100 bootstrap replicates. Trees were

A. Bhunchoth et al. / Virology 494 (2016) 56–66

rooted with the midpoint function of R package phangorn (Schliep, 2011). To reconstruct the viral proteomic tree, we implemented a new bioinformatics pipeline, using a method based on a previous study (Mizuno et al., 2013) with modifications for distance calculation and tree drawing. For every pair of genome x and y (length x olength y) from a set of genomes, we used TBLASTX to identify high scoring segment pairs (HSPs) (Mizuno et al., 2013) to obtain pairwise genome similarity scores, Sxy, where x is the query and y is the subject genome. Length-normalized bit scores (i.e., HSP scores divided by HSP lengths) were then assigned to each base position of genome x. When two or more scores were attributable to a single position, we assigned only the highest normalized score. The genome similarity score, Sxy, was defined as the sum of the length-normalized scores along genome x. Pairwise genome distance, dxy, was then determined (dxy ¼ 1  Sxy/Sxx). The final proteomic tree was generated using BIONJ (Gascuel, 1997) based on dxy. The resulting dendrogram was mid-point rooted for presentation purposes and the branch lengths from the root were scaled logarithmically. In this logarithmic representation, the nodes that were at distances smaller than 0.001 from the root were agglomerated into the root point. Sequence logos were calculated by Weblogo (http://weblogo.berkeley.edu) (Crooks et al., 2004). 4.5. Identification of in-gel digested proteins by liquid chromatography-tandem mass spectrometry Purified phage particles were subjected to SDS-PAGE [10–12% (wt/vol) polyacrylamide] according to the method of Laemmli (1970). Protein bands were visualized with Coomassie Brilliant Blue, excised from the gel, and digested with trypsin after reduction with dithiothreitol and alkylation with iodoacetamide. Tryptic peptides were trapped using a short ODS column (PepMap 100; 5 μm C18, 5 mm  300 μm ID; Thermo Fisher Scientific Inc., Waltham, MA, USA) and then separated with another ODS column (Nano HPLC Capillary Column; 3 μm C18, 120 mm  75 μm ID; Nikkyo Technos, Tokyo, Japan) using nano-liquid chromatography (Ultimate 3000 RSLC nano system; Thermo Fisher Scientific) at the Natural Science Center for Basic Research and Development, Hiroshima University. The mobile phases for separation were solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile). After loading tryptic peptides onto the trap column with 0.1% trifluoroacetic acid for 3 min at a flow rate of 30 ml/min, the concentrated tryptic peptides were eluted from the trap column and separated on the separation column using a sequence of isocratic and linear gradient elution: 0–3 min, solvent A; 3–35 min, 0–35% (v/v) solvent B; increase to 90% solvent B for 10 min; and reequilibrate with solvent A for 15 min. The eluate from the separation column was continuously introduced into a nanoESI source and analyzed by mass spectrometry (MS) and MS/MS (LTQ Orbitrap XL; Thermo Fisher Scientific) at the Natural Science Center for Basic Research and Development, Hiroshima University. The MS and MS/MS spectra were generated in the positive ion mode using Orbitrap (mass range: m/z 300 to 1500) and Iontrap (data-dependent scan of the top five peaks using CID), respectively. The voltage of the capillary source was set at 1.5 kV, and the temperature of the transfer capillary was maintained at 200 °C. The capillary voltage and tube lens voltage were set at 20 V and 80 V, respectively. The assignment of the MS/MS data to tryptic peptides encoded by phage ORFs was completed as previously described (Ahmad et al., 2014) using the Xcalibur program, version 2.0 (Thermo Fisher Scientific). All MS/MS data were searched using Mascot (Matrix Sciences) against the GeneBank non-redundant protein database and against a local database of all possible RSL2 and RSF1 gene products using Proteome Discoverer software (ver. 1.4, Thermo Scientific). Doubly, triply and quadruply charged

65

peptide ions were subjected to the database search with a parent and peptide ion mass tolerance of 710 ppm and 70.8 Da, respectively. Possible static and chemical modifications included were cysteine carbamidomethylation, methionine oxidation and deamidation of asparagine and glutamine. The significance threshold on Proteome Discoverer for Mascot search was set at Po 0.05 and one and two missed trypsin cleavage was allowed. 4.6. In planta virulence assay of R. solanacearum R. solanacearum (strain MAFF 211514; highly virulent to tomato cultivars) was grown in CPG medium for 1–2 d at 28 °C. After centrifugation, the cells were resuspended in distilled water at a cell density of 5  108 cells/ml (OD600 ¼1.0). Soil containing 4week-old tomato plants (Solanum lycopersicum L. cv ‘Oogatafukuju’; with 4–6 leaves) whose roots had been cut with a spatula was inoculated with 5 ml cell suspension. The final concentration of R. solanacearum was approximately 1  106 colony-forming units/g soil. For phage treatments, the phage suspension (5 ml; 2  1010 pfu/ml) was applied to the plants 1 d before bacterial challenge. As a control, distilled water was added in the same manner. Each experiment included five plants and was repeated three times. Plants were cultivated in a Sanyo Growth Cabinet at 25 °C (16 h light/ 8 h dark) for 4 weeks before detailed examination. Symptoms of wilting were graded from 1 to 5 as described by Winstead and Kelman (1952).

Conflict of interest The authors have no conflicts of interest to declare.

Acknowledgments This study was supported by the Strategic Japanese-Thai Research Cooperative Program (SICP) on Biotechnology (JST/BIOTEC-SICP). TY and HO were partially supported by JSPS KAKENHI (Grant numbers 24380049 and 26430184, respectively). The authors would like to thank Guy Schoehn (UMI3265 UJF-EMBLCNRS, France) for providing the electron microscopy images, Takeru Matsui for LC–MS/MS analysis, and Yuya Sugimura and Taiki Yasuda for completing the plant challenge assays. The authors also thank Likhit Maneesinthu for assistance with the tomato field work in Chiang Mai, Thailand. Some of the computational work was completed at the SuperComputer System, Institute for Chemical Research, Kyoto University.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2016.03.028.

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