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. 2018 Jan 31;84(4):e02145-17. doi: 10.1128/AEM.02145-17

Ankyrin-Like Protein AnkB Interacts with CatB, Affects Catalase Activity, and Enhances Resistance of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola to Phenazine-1-Carboxylic Acid

Xiayan Pan a, Shu Xu a, Jian Wu a, Yabing Duan a, Zhitian Zheng a, Jianxin Wang a, Xiushi Song a, Mingguo Zhou a,✉
Editor: Janet L Schottelb
PMCID: PMC5795068  PMID: 29180371

ABSTRACT

Xanthomonas oryzae pv. oryzae, which causes rice bacterial leaf blight, and Xanthomonas oryzae pv. oryzicola, which causes rice bacterial leaf streak, are important plant-pathogenic bacteria. A member of the adaptor protein family, ankyrin protein, has been investigated largely in humans but rarely in plant-pathogenic bacteria. In this study, a novel ankyrin-like protein, AnkB, was identified in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. The expression of ankB was significantly upregulated when these bacteria were treated with phenazine-1-carboxylic acid (PCA). ankB is located 58 bp downstream of the gene catB (which encodes a catalase) in both bacteria, and the gene expression of catB and catalase activity were reduced following ankB deletion in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Furthermore, we demonstrated that AnkB directly interacts with CatB by glutathione S-transferase (GST) pulldown assays. Deletion of ankB increased the sensitivity of X. oryzae pv. oryzae and X. oryzae pv. oryzicola to H2O2 and PCA, decreased bacterial biofilm formation, swimming ability, and exopolysaccharide (EPS) production, and also reduced virulence on rice. Together our results indicate that the ankyrin-like protein AnkB has important and conserved roles in antioxidant systems and pathogenicity in X. oryzae pv. oryzae and X. oryzae pv. oryzicola.

IMPORTANCE This study demonstrates that the ankyrin protein AnkB directly interacts with catalase CatB in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola. Ankyrin protein AnkB can affect the gene expression of catB, catalase activity, and sensitivity to H2O2. In Xanthomonas spp., the locations of genes ankB and catB and the amino acid sequence of AnkB are highly conserved. It is suggested that in prokaryotes, AnkB plays a conserved role in the defense against oxidative stress.

KEYWORDS: ankB, catB, catalase activity, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, phenazine-1-carboxylic acid, oxidative stress

INTRODUCTION

Rice bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae and rice bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola are important diseases of rice worldwide (1, 2). These diseases, which can reduce yields by 75% or more (3–5), can be controlled by phenazine-1-carboxylic acid (PCA), an antibiotic produced by not only Pseudomonas spp. but also plant-associated bacteria, such as Burkholderia, Pectobacterium, Brevibacterium, Streptomyces, and others (6–10). In vitro antibacterial activity of PCA against X. oryzae pv. oryzae and X. oryzae pv. oryzicola has been documented, and PCA has recently been used to control rice bacterial leaf streak in fields in southern China (11–13). In addition to being toxic to bacteria, PCA is also toxic to eukaryotes, as it causes the accumulation of reactive oxygen species (ROS). The mechanism underlying the toxicity of PCA to bacteria is incompletely understood (12, 14). The current study concerns the involvement of an ankyrin-like protein in the responses of X. oryzae pv. oryzae and X. oryzae pv. oryzicola to PCA.

Ankyrins belong to the family of adaptor proteins, which anchor the cytoskeleton to the plasma membrane and thereby provide cellular stability in eukaryotic cells (15, 16). Ankyrins have three domains: an N-terminal membrane-binding domain, a central spectrin-binding domain, and a C-terminal regulatory domain (17, 18). Ankyrins are widespread in eukaryotes but are uncommon in prokaryotes (19). Most research concerning ankyrins has been conducted in humans because a variety of human diseases are related to the dysfunction of ankyrin proteins (20). Functional analysis confirmed that ankyrins have a regulatory or structural role rather than an enzymatic one (21). Ankyrins in plants are involved in defense responses to ROS and in regulating the hypersensitive reaction (HR) (22). Overexpression of the ankyrin repeat-containing protein OsBIANK1 in Arabidopsis decreases ROS levels after infection by Botrytis cinerea (23). Ankyrins are also necessary for regulating cell motility, adhesion, and the maintenance of specialized membrane domains, ion channels, and transporters (20, 24, 25). They are also involved in intracellular signaling, such as regulating the transcription factor NF-κB to influence gene expression (26). Together, these studies demonstrate that ankyrins have a variety of functions.

In bacteria, genes encoding predicted ankyrin-like proteins are often located in close proximity to genes encoding proteins involved in responses to oxidative stress (27–29). The Cj1386 gene, encoding an ankyrin-containing protein, is located downstream from katA (which encodes a catalase) and is involved in the same detoxification pathway as catalase in Campylobacter jejuni (30). In Pseudomonas aeruginosa, the ankyrin AnkB is required for the detoxification of H2O2 by catalase (KatB) (29). In our previous study, CatB was found to be the key protein for total catalase activity and reduced bactericidal effects of PCA on X. oryzae pv. oryzae and X. oryzae pv. oryzicola (31). Similarly, the gene encoding the ankyrin protein AnkB is also located downstream from the gene encoding CatB (catalase) in these two strains. The ankyrins and their functions in plant-pathogenic bacteria have not been characterized. In addition, there are no reports concerning interactions between ankyrins and catalases in any other species. Therefore, investigation to resolve whether AnkB is required for catalase activity in X. oryzae pv. oryzae and X. oryzae pv. oryzicola in response to PCA and, if so, whether the two proteins directly interact is warranted.

In this study, we cloned the gene ankB, which encodes an ankyrin-like protein in X. oryzae pv. oryzae and X. oryzae pv. oryzicola, and investigated the role of AnkB when the two bacteria are under oxidative stress. Our study also revealed the relationship between AnkB and CatB and the functions of AnkB involved in protecting cells from PCA and regulating the virulence of both bacteria.

RESULTS

ankB expression is upregulated in X. oryzae pv. oryzae and X. oryzae pv. oryzicola by exogenous PCA.

Transcriptome sequencing (RNA-seq) showed that the expression of ankB was strongly increased in X. oryzae pv. oryzae ZJ173 in response to treatment with 4 μg/ml PCA (data not shown). The highest PCA concentrations that did not slow culture growth were determined to be 4 μg/ml for ZJ173 and 32 μg/ml for RS105. The quantitative reverse transcription-PCR (qRT-PCR) experiment was repeated to verify the results. The results revealed that the expression of ankB in X. oryzae pv. oryzae ZJ173 was 3.5-, 7.5-, and 11.4-fold upregulated in response to PCA at 1, 2, and 4 μg/ml PCA, respectively (Fig. 1A). Similarly, the expression of ankB in X. oryzae pv. oryzicola RS105 was 3.6-, 3.1-, and 8.1-fold upregulated in response to PCA at 8, 16, and 32 μg/ml PCA, respectively (Fig. 1B).

FIG 1.

FIG 1

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(A and B) Relative expression levels of ankB in Xanthomonas oryzae pv. oryzae (strain ZJ173) (A) and Xanthomonas oryzae pv. oryzicola (strain RS105) (B) when stimulated with PCA, determined by RT-PCR. The tested strains were grown in NB medium to an OD600 of 0.2, and PCA was added to final concentrations of 1, 2, and 4 μg/ml for ZJ173 and 8, 16, and 32 μg/ml for RS105. The amount of RNA in acetone was used as the control and was set at 1.0. Values are means ± standard deviations (SD) from three technical replicates. Similar results were obtained from three biological replicates. *, P < 0.05 (Student's t test). (C) GenBank accession numbers are according to KEGG. The red lines represent the locations of primers in real-time RT-PCR. →, forward primer; ←, reverse primer (for qRT-PCR). The arrows in purple and blue represent the starting and end positions of the deletion of ankB, respectively. Xoo, Xanthomonas oryzae pv. oryzae; Xoc, Xanthomonas oryzae pv. oryzicola. (D) The strains were Xanthomonas oryzae pv. oryzae (strain KACC 10331), Xanthomonas oryzae pv. oryzicola (strain BLS256), Xanthomonas axonopodis pv. vasculorum (Xav), Xanthomonas citri pv. citri (Xac; strain 306), Xanthomonas campestris pv. campestris (Xcc; strain ATCC 33913), Xanthomonas campestris pv. raphani (Xcp; strain 756C), and Xanthomonas citri subsp. citri (Xcr; strain MN11). Amino acid sites that differ are in color on a white background. (E) Growth rates of wild-type strains, ankB deletion mutants, and their complemented mutants of Xanthomonas oryzae pv. oryzae ZJ173 and Xanthomonas oryzae pv. oryzicola RS105 in a nutrient-rich medium (NB) as indicated by optical density at 600 nm (OD600).

Sequence analysis, deletion, and complementation of ankB in X. oryzae pv. oryzae and X. oryzae pv. oryzicola.

ankB in X. oryzae pv. oryzae (XOO0418) and ankB in X. oryzae pv. oryzicola (XOC_4324) (gene names are according to the annotations of X. oryzae pv. oryzae KACC 10331 and X. oryzae pv. oryzicola BLS256, respectively) are previously uncharacterized genes that encode ankyrin-like proteins. The ankB nucleotide sequences in these two strains are 576 bp long and encode 191 amino acids, which include ankyrin repeats. ankB is located 58 bp downstream of catB (which encodes a catalase) in both strains (Fig. 1C) (http://www.genome.jp/kegg/). According to the NCBI database, the sequence from bp 69 to 175 of ankB of X. oryzae pv. oryzae KACC 10331 is an ANK superfamily domain (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi?INPUT_TYPE=precalc&SEQUENCE=58424635). By analyzing the sequence alignment in ANK superfamily domains, we found that the ankyrin repeat domains are highly conserved in Xanthomonas (Fig. 1D).

To investigate the role of ankB in X. oryzae pv. oryzae and X. oryzae pv. oryzicola, we generated targeted deletion mutants of ZJ173 and RS105 by a nonmarker homologous recombination method. Deletion strains ΔZ (ankB) of X. oryzae pv. oryzae ZJ173 and ΔR (ankB) of X. oryzae pv. oryzicola RS105 were verified by PCR using the ZankB-1F/2R and RankB-1F/2R primers (Table 1), respectively. qRT-PCR analysis showed that ankB expression was absent in the deletion mutants (Fig. 2). The ΔZ (ankB) mutant and the ΔR (ankB) mutant were complemented with plasmids pUFRZCB and pUFRRCB, which are derivatives of pUFR034 carrying the open reading frames of ankB and their predicted promoters in ZJ173 or RS105, respectively. The promoters were 123 bp upstream of the start codon of the ankB coding sequence in both X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Complementation of the mutants was confirmed by PCR (see Fig. S1C in the supplemental material).

TABLE 1.

Strains, plasmids, and primers used in this study

Strain, plasmid, or primer Characteristics or sequence Reference or source
Strains
    Escherichia coli DH5α ϕ80 lacZΔM15 Δ(lacZYA-argF)U169 recA1 Vazyme, Nanjing, China
    Xanthomonas oryzae pv. oryzae
        ZJ173 Rifr; wild-type strain 12
        ΔZ (ankB) Rifr; deletion of ankB in wild-type strain ZJ173 This study
        ΔZ (ankB)/ankB Rifr Kmr; complemented strain of ΔZ (ankB) This study
        ΔZ (catB) Rifr; deletion of catB in wild-type strain ZJ173 31
        ΔZ (catB)/catB Rifr Kmr; complemented strain of ΔZ (catB) 31
    Xanthomonas oryzae pv. oryzicola
        RS105 Rifr; wild-type strain 12
        ΔR (ankB) Rifr; deletion of ankB in wild-type strain RS105 This study
        ΔR (ankB)/ankB Rifr Kmr; complemented strain of ΔR (ankB) This study
        ΔR (catB) Rifr; deletion of catB in wild-type strain RS105 31
        ΔR (catB)/catB Rifr Kmr; complemented strain of ΔR (catB) 31
Plasmids
    pK18mobsacB Kmr; allelic exchange suicide vector, sacB oriT(RP4) 54
    pK18ZAB Kmr; 1,839-bp fusion fragment of ZankB-1, ZankB-2 gene ligated in pK18mobsacB This study
    pK18RAB Kmr; 1,796-bp fusion fragment of RankB-1, RankB-2 gene ligated in pK18mobsacB This study
    pUFR034 IncW; Nmr Kmr; mob+ mob(p) lacZα, PK2 replicon, cosmid 55
    pUFRZCB Kmr; 755-bp fusion fragment of ZankB-H gene in ZJ173 ligated in pUFR034 This study
    pUFRRCB Kmr; 755-bp fusion fragment of RankB-H gene in RS105 ligated in pUFR034 This study
    pGEX-2TK Ampr Cmr; tac, expression of GST fusion protein 62
    pGEX-ZcatB Kmr; 1,524-bp fusion fragment of ZcatB gene in ZJ173 ligated in pGEX-2TK This study
    pGEX-RcatB Kmr; 1,524-bp fusion fragment of RankB gene in RS105 ligated in pGEX-2TK This study
    pET-28a Kmr; T7, expression of His6 fusion protein 63
    pET-ZcatB Kmr; 576-bp fusion fragment of ZankB gene in ZJ173 ligated in pET-28a This study
    pET-RcatB Kmr; 576-bp fusion fragment of RankB gene in RS105 ligated in pET-28a This study
Primers
    ZankB-1F 5′-CGGGATCCAAGAATCTCGATCCCAAAC-3′ This study
    ZankB-1R 5′-CGGAATTCAGACAGGCTGCCAAGC-3′ This study
    ZankB-2F 5′-CGGAATTCGCATGCTGCTCTTGCTTG-3′ This study
    ZankB-2R 5′-GCTCTAGAGGGTTACGGACACCCACA-3′ This study
    RankB-1F 5′-CGGGATCCGAATCTCGATCCCAAACA-3′ This study
    RankB-1R 5′-CGGAATTCGAATCTCCCGGACTCAA-3′ This study
    RankB-2F 5′-CGGAATTCGCATGCTGCTCTTGCTTG-3′ This study
    RankB-2R 5′-GCTCTAGAGGTTACGGACACCCACA-3′ This study
    Z/RankBH-F 5′-GGGGTACCAAGCAAGCACCGGATGCT-3′ This study
    Z/RankBH-R 5′-CGGGATCCTCAGCGCGCCGGGGCGGT-3′ This study
    GST-Z/RcatB-F 5′-CGGGATCCATGCGCCCTGGATCTCTC-3′ This study
    GST-Z/RcatB-R 5′-TCCCCCGGGTCAGTCCTGCAGGCTGGA-3′ This study
    His-Z/RankB-F 5′-CGGGATCCATGCGCACCCTCTTGTTT-3′ This study
    His-Z/RankB-R 5′-CCGCTCGAGTCAGCGCGCCGGGGCGGT-3′ This study
    Z16S rRNA-F 5′-GGCGAGCACAATGGCATT-3′ 12
    Z16S rRNA-R 5′-CCATCCTTCTGCGGGATGT-3′ 12
    R16S rRNA-F 5′-AATGGGCGCAAGCCTGATC-3′ 53
    R16S rRNA-R 5′-AACCACCACCTACGCACGC-3′ 53
    qZcatB-F 5′-CGCCCAATCCGTTCTGA-3′ 31
    qZcatB-R 5′-CGGTGAACTCGCCTTTGA-3′ 31
    qZankB-F 5′-GTGAAGGTCGCCAAGACAT-3′ This study
    qZankB-R 5′-CCAGGATCAACGCGGTATAG-3′ This study
    qRcatB-F 5′-CAGGGCCGTATCTTCTCTTATG-3′ 31
    qRcatB-R 5′-ATCCTGGTTGCCGTTGTT-3′ 31
    qRankB-F 5′-CACACCCGAGCAGATCAAG-3′ This study
    qRankB-R 5′-CGTAGTGCGAGCGAATGAA-3′ This study
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FIG 2.

FIG 2

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Gene expression (A and B) and total catalase activity (C and D) of ankB and catB in Xanthomonas oryzae pv. oryzae ZJ173 (A and C) and Xanthomonas oryzae pv. oryzicola (B and D) and their ankB deletion and complemented mutants. The tested strains were grown in NB medium. (A and B) Gene expression was detected by real-time RT-PCR. Relative mRNA levels of ankB and catB in ZJ173 and RS105 were set at 1. Values are means ± SD from three biological replicates. *, P < 0.05 (t test). (C and D) Total catalase activity was measured from total protein extract. Values are means ± SD from three biological replicates. *, P < 0.05 (t test).

ankB is necessary for expression of catB and for total catalase activity in X. oryzae pv. oryzae and X. oryzae pv. oryzicola under no stress or PCA stress.

To investigate the importance of ankB in affecting expression of the catalase gene catB, we analyzed the expression of catB in the ankB deletion mutants. The gene expression of catB in X. oryzae pv. oryzae and X. oryzae pv. oryzicola was significantly reduced in ΔZ (ankB) and ΔR (ankB) but was restored in the complemented strains (Fig. 2A and B). In addition, catalase activity was significantly lower in ΔZ (ankB) than in the wild-type strain ZJ173 (Fig. 2C). Catalase activity was also significantly lower in ΔR (ankB) than in the wild-type strain RS105 (Fig. 2D). As expected, catalase activities did not significantly differ between the complemented mutants ΔZ (ankB)/ankB and ΔR (ankB)/ankB and their corresponding wild-type strains.

To further confirm that AnkB affects CatB activity, qRT-PCR was used to investigate the expression of catB in response to PCA stress in ankB deletion mutants of X. oryzae pv. oryzae and X. oryzae pv. oryzicola. The results demonstrated that the gene expression of catB was significantly induced after treatment with PCA at 4 μg/ml for X. oryzae pv. oryzae and at 32 μg/ml for X. oryzae pv. oryzicola (Fig. 3A and B). However, the upregulation of catB was much lower in the deletion mutants ΔZ (ankB) and ΔR (ankB) than in the wild-type strains (Fig. 3B). Catalase activities in ΔZ (ankB) and ΔR (ankB) were also significantly reduced by PCA (Fig. 3C).

FIG 3.

FIG 3

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(A and B) Gene expression of ankB (A) and catB (B) in Xanthomonas oryzae pv. oryzae ZJ173 and Xanthomonas oryzae pv. oryzicola RS015 and their ankB deletion and complemented mutants when treated with 4 and 32 μg/ml PCA, respectively. (C) Total catalase activity of ankB deletion mutants of ZJ173 and RS105 when treated with 4 and 32 μg/ml PCA, respectively. In all cases, strains were grown and treated in NB medium; values are means ± SD from three biological replicates. *, P < 0.05 (t test). For panels A and B, expression was assessed by real-time RT-PCR. Relative mRNA levels of ankB and catB in ZJ173 and RS105 when treated with acetone were set at 1. For panel C, total catalase activity was measured from total protein extract.

AnkB directly interacts with CatB and enhances the resistance of X. oryzae pv. oryzae and X. oryzae pv. oryzicola to H2O2 and PCA.

Having established that AnkB likely functions as a protein interacting with CatB, we then used glutathione S-transferase (GST) pulldown to verify our speculation. AnkB-His was detected when CatB-GST was present in the mixture in both X. oryzae pv. oryzae and X. oryzae pv. oryzicola, indicating that AnkB can directly interact with CatB in vitro (Fig. 4A).

FIG 4.

FIG 4

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(A) Interaction between AnkB and CatB as determined by a GST pulldown assay. The purified GST-CatB was incubated with AnkB-His6 and pulled down with GST beads. GST-CatB pulled down a significant amount of AnkB-His6 (bands marked with asterisks) by immunoblotting using anti-His antibody. The numbers represent the molecule sizes of protein markers. (B) H2O2 sensitivity of Xanthomonas oryzae pv. oryzae ZJ173, Xanthomonas oryzae pv. oryzicola RS015, and their ankB deletion and complemented mutants on NB medium plates. One-, 3-, and 9-fold dilutions (1×, 3×, and 9×) of the bacterial suspension were added to NB medium containing 0, 0.05, 0.1, or 0.2 mM H2O2.

To further investigate the role of ankB in the detoxification of exogenous ROS, an H2O2 growth inhibition assay was carried out with ΔZ (ankB) and ΔR (ankB). As expected, ΔZ (ankB) and ΔR (ankB) were more susceptible to H2O2 than their wild-type strains (Fig. 4B). Susceptibility to H2O2 did not significantly differ, however, between the complemented strains and wild-type strains. In a PCA growth inhibition assay, we found that the sensitivity to PCA was much higher in ΔZ (ankB) and ΔR (ankB) than in the wild-type strains and complemented strains (Table 2).

TABLE 2.

Sensitivities of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola strains to phenazine-1-carboxylic acid

graphic file with name zam00418-8306-t02.jpg

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a

Values are means ± standard errors from three experiments. Values followed by the same letter are not significantly different (P > 0.05).

ankB is essential for X. oryzae pv. oryzae and X. oryzae pv. oryzicola biofilm formation, swimming motility, EPS production, and virulence on rice.

Since the ankyrin protein AnkB was related to oxidative stress, which influences most biological processes of cells, we evaluated the roles of AnkB in biofilm formation, swimming motility, exopolysaccharide (EPS) production, and virulence on rice. A biofilm formation assay showed that the mass of biofilm formed was 55% lower for the ΔZ (ankB) and ΔZ (catB) strains than for the wild-type strain ZJ173. Similarly, biofilm formation was significantly lower for strains ΔR (ankB) and ΔR (catB) than for the wild-type strain RS105 (Fig. 5A). In the swimming ability assay, strains ΔZ (ankB) and ΔZ (catB) formed smaller colonies with less diffuse surfaces than the wild-type strain ZJ173 on a semisolid nutrient agar (NA) plate. Colony diameter and morphology also indicated that the ability to swim was reduced for strains ΔR (ankB) and ΔR (catB) relative to the wild-type strain RS105 (Fig. 5B). In the EPS production assay, strains ΔZ (ankB) and ΔZ (catB) produced smaller amounts of EPS (1.09 and 0.67 mg/ml, respectively) than the wild-type strain ZJ173 (1.75 mg/ml) in NB medium. EPS production also decreased in strains ΔR (ankB) and ΔR (catB) (0.53 and 0.46 mg/ml, respectively) compared with the wild-type strain RS105 (1.30 mg/ml) (Fig. 5C). These decreases in biofilm formation, swimming motility, and EPS production were all rescued in the complemented strains ΔZ (ankB)/ankB and ΔR (ankB)/ankB (Fig. 5).

FIG 5.

FIG 5

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Biofilm formation (A), swimming ability (B), and EPS production (C) of Xanthomonas oryzae pv. oryzae ZJ173, Xanthomonas oryzae pv. oryzicola RS015, and their ankB deletion and complemented mutants. Representative biological phenotypes were photographed. Values are means ± SD from three biological replicates, which had similar results. *, P < 0.01 (Student's t test).

To assess the importance of ankB for X. oryzae pv. oryzae and X. oryzae pv. oryzicola virulence on rice, we inoculated the susceptible rice cultivar IR24 with X. oryzae pv. oryzae ZJ173, ΔZ (ankB), ΔZ (ankB)/ankB, X. oryzae pv. oryzicola RS105, ΔR (ankB), and ΔR (ankB)/ankB. The lesions were much shorter for strains ΔZ (ankB) and ΔR (ankB) (1.8 ± 0.4 and 1.3 ± 0.2 cm, respectively) than for strains ZJ173 and RS105 (5.5 ± 0.4 and 4.3 ± 0.3 cm, respectively) (Fig. 6). Lesion length was more than 90% restored in the ankB complemented strains.

FIG 6.

FIG 6

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Pathogenicity of Xanthomonas oryzae pv. oryzae strains (A) and Xanthomonas oryzae pv. oryzicola strains (B) in rice cultivar IR24. The bar charts show lesion lengths. Three replicates were used for each treatment, and the experiment was repeated two times. Values are the means ± SD from three replicates.

DISCUSSION

Previous studies demonstrated that ankyrins have essential regulatory and structural functions in eukaryotes and viruses (20, 32–35). However, the functions of ankyrins in prokaryotes are largely unknown. Our study revealed that ankB, encoding the ankyrin AnkB in X. oryzae pv. oryzae and X. oryzae pv. oryzicola, affects the gene expression of catB and catalase activity. Moreover, AnkB directly interacts with CatB and enhances resistance to H2O2 and PCA. Our study also revealed that AnkB participates in X. oryzae pv. oryzae and X. oryzae pv. oryzicola biofilm formation, swimming ability, EPS production, and pathogenicity.

Earlier studies revealed that PCA functions as a redox-active compound that generates ROS not only in X. oryzae pv. oryzae but also in Vibrio anguillarum and Phellinus noxius (8, 12, 31, 36–38). The enhanced ROS production can lead to altered electrical charge and to DNA or membrane damage in cells (39, 40). Catalase is one of the most important enzymatic mechanisms to detoxify excessive levels of ROS (41). We previously reported that the expression of the catalase-encoding genes catB and katE in X. oryzae pv. oryzae and X. oryzae pv. oryzicola increased dramatically in response to PCA treatment (31). In this study, the gene expression of ankB in X. oryzae pv. oryzae and X. oryzae pv. oryzicola was also significantly increased when bacteria were treated with PCA, indicating that the protein encoded by ankB (AnkB) may be important for bacterial resistance to PCA (Fig. 1A and B). In Pseudomonas aeruginosa, AnkB stabilizes KatB, and the location of ankB is 57 bp downstream of katB. Intriguingly, the location of these genes is similar (ankB is 58 bp downstream of catB) in X. oryzae pv. oryzae and X. oryzae pv. oryzicola, suggesting that the ankyrin protein AnkB is closely linked to the catalase CatB in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. The alignment analysis showed that the amino acid sequence of the ANK superfamily is highly conserved in Xanthomonas spp. (Fig. 1D), suggesting that the function of ankyrins is relatively conserved.

The growth rate experiment showed that the growth rates of the deletion mutants did not differ from those of the wild-type strains in NB medium (Fig. 1E). Therefore, we used NB medium in the experiments to ensure that the differences exhibited by the ankB deletion mutants were not caused by growth defects. We found that catB gene expression and catalase activities were significantly decreased in the ankB deletion mutants of X. oryzae pv. oryzae and X. oryzae pv. oryzicola (Fig. 2), indicating that the ankyrin protein AnkB is important for gene expression of catB and for catalase activity in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. In line with our findings and as noted earlier, AnkB is required for catalase (KatB) activity in Pseudomonas aeruginosa (29). Furthermore, the expression of catB in ankB deletion mutants of X. oryzae pv. oryzae and X. oryzae pv. oryzicola was partly activated in response to PCA, and catalase activities in both deletion mutants were significantly inhibited by PCA (Fig. 3). Combined with a previous report that PCA acts as a redox-active compound and leads to ROS accumulation in X. oryzae pv. oryzae (12), the current results further demonstrate that the ankyrin protein AnkB alters catalase function under oxidative stress. In view of these results, we hypothesized that AnkB interacts with CatB to ensure full catalase activity in response to PCA. Consistent with our hypothesis, a GST pulldown assay showed that AnkB directly interacts with CatB in X. oryzae pv. oryzae and X. oryzae pv. oryzicola (Fig. 4A). Because CatB helped protect X. oryzae pv. oryzae and X. oryzae pv. oryzicola under H2O2 and PCA stress (42), we investigated the antioxidation activity in the ankB deletion mutants of X. oryzae pv. oryzae and X. oryzae pv. oryzicola under H2O2 and PCA stress. We found that the ankB deletion mutants were more sensitive to H2O2 and PCA than the wild-type strains (Fig. 4B; Table. 2), indicating that AnkB is essential for protecting X. oryzae pv. oryzae and X. oryzae pv. oryzicola against H2O2 and PCA. These results further suggested that catalase protection of cells against oxidizing agents in X. oryzae pv. oryzae and X. oryzae pv. oryzicola partly depends on the ankyrin protein AnkB.

Recent studies found that oxidative stress in Campylobacter jejuni and KatG (catalase) in Xanthomonas citri subsp. citri are important in biofilm formation (43, 44). In an antioxidase ahpC mutant of Vibrio parahaemolyticus, the ability to swim in a semisolid medium was decreased (45). It also was reported that EPS had an important role in protecting cells against ROS in plant-pathogenic bacteria (46). Consistent with these previous studies, our results clearly indicated that catalase and ankyrin protein are necessary for biofilm formation, swimming ability, and EPS production in X. oryzae pv. oryzae and X. oryzae pv. oryzicola, because these phenotypes were reduced in catB and ankB gene deletion mutants of X. oryzae pv. oryzae ZJ173 and X. oryzae pv. oryzicola RS105 (Fig. 5). Many studies showed that ROS and their detoxifying enzymes are involved in plant defense (47–50). Catalases were required for plant pathogenesis in Pseudomonas syringae and Xanthomonas campestris pv. campestris (51, 52). In our study, virulence was lower for strains ΔZ (ankB) and ΔR (ankB) than for their wild-type strains (Fig. 6). Given that catB deletion mutants of X. oryzae pv. oryzae and X. oryzae pv. oryzicola had reduced virulence (31, 42), we speculate that the reduced catalase activity in ankB deletion mutants reduces the ability of the bacteria to detoxify ROS and therefore reduces virulence. Although the results have revealed that catB and ankB are involved in biofilm formation, swimming ability, EPS production, and pathogenicity, the phenotypes were less severe for the ankB mutants than for the catB mutants. This may be attributed to the possibility that the ankyrin AnkB only indirectly affects physiological functions by affecting the function of catalase CatB.

In summary, our study demonstrates a direct interaction between the ankyrin AnkB and the catalase CatB. It further provides evidence that deletion of ankB results in lower gene expression of catB and catalase activity, which in turn increases PCA sensitivity in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. AnkB is also important for biofilm formation, swimming ability, EPS production, and virulence in X. oryzae pv. oryzae and X. oryzae pv. oryzicola.

MATERIALS AND METHODS

Strains, plasmids, bactericides, and media.

All strains and plasmids used in this study are listed in Table 1. ZJ173 and RS105, the wild-type strains of X. oryzae pv. oryzae and X. oryzae pv. oryzicola used in this study, respectively, are commonly used in China (12). X. oryzae pv. oryzae and X. oryzae pv. oryzicola strains were grown at 28°C in nutrient broth (NB) medium, consisting of 1 μg/ml yeast extract, 3 μg/ml beef extract, 5 μg/ml polypeptone, and 10 μg/ml sucrose. Nutrient agar (NA) medium contained the same components plus 12 μg/ml agar powder. Minimal (MMX) medium at pH 7.0 contained 0.2 μg/ml MgSO4 · 7H2O, 5 μg/ml glucose, 2 μg/ml (NH4)2SO4, 4 μg/ml K2HPO4, 6 μg/ml KH2PO4, and 1 μg/ml trisodium citrate. Escherichia coli strains were grown at 37°C. E. coli DH5α (Vazyme), which was used in vector construction, was cultured in LB medium containing 50 μg/ml kanamycin. E. coli Rosetta (Tiangen Biotech, Beijing, China) was used in protein expression and was cultured in LB medium containing 100 μg/ml ampicillin, 20 μg/ml gentamicin, and 50 μg/ml of kanamycin. Phenazine-1-carboxylic acid (PCA) (98%) was provided by Shanghai Nongle Biological Products Co., Ltd. (China) and was dissolved in acetone as a stock solution.

Determination of gene expression of ankB and catB in X. oryzae pv. oryzae and X. oryzae pv. oryzicola.

Gene expression of ankB and catB in ZJ173 and RS105 was detected with real-time PCR. Before doing the qRT-PCR experiment, we did a preliminary experiment with a concentration gradient of PCA and selected the concentration at which the gene expression changed most obviously but growth was not inhibited. For ZJ173 this was 4 μg/ml, and for RS105 it was 32 μg/ml (as well as for the following experiments performed in this study). To perform qRT-PCR, all tested strains were grown to early logarithmic phase at an optical density at 600 nm (OD600) of 0.2 (2 × 108 CFU/ml) in NB medium at 28°C with shaking at 175 rpm. A 25-ml culture was then treated with 100 μl of different concentrations of PCA. After incubating for approximately 4 h when the OD600 was ≈0.5 (5 × 108 CFU/ml), the cultures were harvested by centrifugation (12,000 × g for 2 min at 4°C). Total RNA was extracted with the RNAprep pure cell/bacteria kit (Tiangen Biotech, Beijing, China) and reverse transcribed into cDNA with the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, Nanjing, China) according to the instruction manual. qRT-PCR was conducted using SYBR qPCR master mix (Vazyme, Nanjing, China) with a CFX Connect real-time system (Bio-Rad, CA, USA). Gene expression of ankB and catB in X. oryzae pv. oryzae (XOO0418 [ankB] and XOO0417 [catB]) and X. oryzae pv. oryzicola (XOC_4324 [ankB] and XOC_4325 [catB]) was assessed, and 16S rRNA was used as the internal control. The primers are listed in Table 1. Three biological replicates were conducted in this experiment, each repeated three times.

Generation of ankB deletion mutants in X. oryzae pv. oryzae and X. oryzae pv. oryzicola.

We used a nonmarker homologous recombination method (53) to generate the deletion mutants in order to investigate the functions of AnkB in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. The suicide vector pK18mobsacB was used in this study (54). The whole-genome sequences of KACC 10331 and BLS256 were used as reference sequences for ZJ173 and RS105, respectively. The genomic DNAs of ZJ173 and RS105 were used as the templates to amplify the upstream and downstream fragments of ankB. The primers for all upstream and downstream fragments are listed in Table 1. ZJ173 and RS105 competent cells were transformed with the recombinant plasmids listed in Table 1 using an electroporation method (55), and the cells were subsequently screened on NAN (NA medium without sucrose) plates containing 20 μg/ml kanamycin. Single colonies were picked and added to 25 ml of NBN medium (NB medium without sucrose). After 7 to 9 h of incubation at 28°C, 120 μl of the suspension was spread on NAS (NA medium containing 100 μg/ml sucrose) plates. The constructs were subsequently confirmed by PCR and qRT-PCR using the primers listed in Table 1. The confirmed mutants were used in further studies. Complemented mutants were constructed using plasmid pUFR034. The whole sequences of ankB (including predicted promoters) were amplified from genomic DNAs of ZJ173 and RS105 with the primers listed in Table 1. The prediction of promoter sequences and electroporation methods were previously described (56).

Determination of growth rate in NB medium.

All tested strains were cultured to an OD600 of 1.0 (109 CFU/ml) in NB medium at 28°C with shaking at 175 rpm. Bacterial cells were then collected by centrifugation (4,000 × g for 2 min) from 2 ml of culture. The cells were resuspended in 2 ml of fresh NB medium. A 2-ml volume of cell resuspension of the X. oryzae pv. oryzae wild-type strain ZJ173 and its ankB mutant was added to 100 ml of NB medium. A 1.2-ml volume of the cell resuspension of X. oryzae pv. oryzicola wild-type strain RS105 and its ankB mutant was added to 100 ml of NB medium. The cultures were then grown at 28°C with shaking at 175 rpm. The OD600 values were determined using an Eppendorf BioPhotometer Plus every 2 h during 12 h of incubation.

Determination of catalase activity.

All tested strains were cultured to an OD600 of 0.5 (5 × 108 CFU/ml) in NB medium at 28°C with shaking at 175 rpm. Bacterial cells were then collected from 2 ml of culture by centrifugation (8,000 × g for 2 min at 4°C), and were resuspended in 200 μl of Western and IP cell lysis liquid (Beyotime, China). The supernatant was collected by centrifugation (8,000 × g for 2 min at 4°C) after 30 min of incubation. The protein concentrations were determined with the bicinchoninic acid (BCA) protein concentration determination kit (Beyotime, China). Total catalase activities were then determined with a catalase test kit (Beyotime, China). Each sample was assayed three times, and three independent experiments were carried out. The results were analyzed with SPSS 20.0 (independent-sample t tests).

Expression and purification of proteins.

The full-length ankB and catB genes of X. oryzae pv. oryzae ZJ173 and X. oryzae pv. oryzicola RS105 were amplified with the primers shown in Table 1. catB was ligated into vector pGEX-2TK for GST-tagged protein expression, and ankB was ligated into vector pET-28a for His-tagged protein expression. All recombinant vectors were used to transform the E. coli Rosetta strain, and cells were collected by centrifugation (5,000 × g for 15 min at 4°C) after induction for 12 to 16 h in LB medium containing 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside). The cells were resuspended in 15 ml of 1× extraction buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol [DTT]) and were subjected to ultrasonication after addition of 40 μl of lysozyme. The precipitate was removed by centrifugation (10,000 × g for 60 min at 4°C), and the supernatant was stored at −80°C.

GST pulldown assay and Western blotting.

The GST-labeled proteins were incubated with glutathione-Sepharose beads (Beyotime, China). The proteins retained by the glutathione-Sepharose beads were mixed with 900 μl of His6-tagged proteins. Bound proteins were analyzed by SDS-PAGE and detected by Western analysis using a previously reported method (57). Briefly, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane that had been soaked in methanol for 3 to 5 min. The membrane was then placed in 5% skim milk for 1 h before primary anti-GST (1:8,000; Sigma-Aldrich) or anti-His (1:5,000; Sigma-Aldrich) antibodies were added to probe the resulting blots. The preparation was kept at room temperature for 90 min, and then horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:10,000; Sigma-Aldrich) or anti-mouse (1:10,000; Sigma-Aldrich) secondary antibodies were added and incubated at room temperature for 60 min for detection. Finally, the resulting blots were developed using the ECL substrate kit (Thermo Scientific, USA).

Determination of H2O2 sensitivity.

All tested strains were cultured to an OD600 of 1.0 (109 CFU/ml) in NB medium, and suspensions were diluted 3-fold and 9-fold in NB medium. NA plates containing 0, 0.05, 0.1, or 0.2 mM H2O2 were also prepared. A 5-μl volume of undiluted or diluted culture was spotted onto NA plates (in triplicate), and the plates were kept for 48 h at 28°C (31). H2O2 sensitivity was assessed based on colony growth. This experiment was repeated three times.

Determination of PCA sensitivity.

All tested strains were grown to an OD600 of 1.0 (109 CFU/ml) at 28°C with shaking at 175 rpm in NB medium, and the bacterial suspensions were adjusted to an OD600 of 0.2 with fresh NB medium. A 120-μl volume of the diluted bacterial suspension was then added to 25 ml of fresh NB medium containing PCA at 0, 0.025, 0.05, 0.1, 0.2, 0.4, or 0.8 μg/ml for the X. oryzae pv. oryzae suspension or at 0, 1, 2, 4, 8, 16, or 32 μg/ml for the X. oryzae pv. oryzicola suspension; the final acetone concentration was 0.4% (vol/vol) in all cases. Three replicates for each PCA concentration were used for each strain. Bacterial cultures were grown at 28°C with shaking at 175 rpm for 36 h for X. oryzae pv. oryzae and for 24 h for X. oryzae pv. oryzicola. For each strain, the average OD600 values were used to calculate the PCA concentration that resulted in 50% inhibition of bacterial cell growth (EC50). The EC50s were calculated with the Data Processing System (DPS) computer program (Hangzhou Reifeng Information Technology Ltd., Hangzhou, China). Each sample was assayed three times, and the experiment was repeated three times.

Biofilm formation, swimming ability, and EPS production assays.

For biofilm formation, bacterial strains were grown in NB medium at 28°C with shaking at 175 rpm until an OD600 of 0.6 (6 × 108 CFU/ml) was attained. A 50-μl volume of each culture was transferred to a glass tube containing 2 ml of fresh NB medium, and the tubes were kept at 28°C. The cultures were removed after 5 days for X. oryzae pv. oryzae and 3 days for X. oryzae pv. oryzicola. Two milliliters of 1% (wt/vol) crystal violet was added to each tube, and the tubes were incubated for 15 min at room temperature. The unbound dye was removed by washing with water, and the tubes were air dried and then photographed. For quantification, the bound dyes were dissolved in ethanol, and the OD590 of the sample was measured. Each strain was assayed three time, and the experiment was repeated three times.

To assay swimming ability, bacterial strains were grown in NB medium at 28°C with shaking (175 rpm) until an OD600 of 1.0 (109 CFU/ml) was attained. Cells were collected by centrifugation and were resuspended in water. A 2-μl volume of each suspension was added to swim plates (peptone, 0.3 g/liter; yeast extract, 0.3 g/liter; agar, 2.5 g/liter) (58), and the plates were kept at 28°C. The swimming ability zone as indicated by colony morphology was measured after 3 days for X. oryzae pv. oryzae and after 2 days for X. oryzae pv. oryzicola. For EPS production, a 500-μl volume of each cell suspension was added to 25 ml of fresh NB medium and then cultured for 5 days at 28°C in shaking flasks (175 rpm). The culture densities of all test strains were normalized because the initial concentration of bacteria was equal and the growth rate in NB medium was also the same. The bacterial cells were harvested by centrifugation (8,000 rpm, 10 min), and a 3-fold volume of 95% ethanol was added to the supernatant. The supernatant was stored overnight and then centrifuged (8,000 rpm, 10 min) again (59). The pellet, which consisted of EPS, was dried at 60°C to a constant weight (60). Carbohydrates were not quantified in medium controls since the volume of NB medium was the same (25 ml) for every test strain. Each EPS assay was replicated three times, and the experiments were repeated three times.

Pathogenicity assay.

The susceptible rice cultivar IR24 was used for pathogenicity assays, which were conducted in a lighted growth chamber at 25°C. The rice cultivar was inoculated with bacterial suspensions as previously described (61) with slight modification. In brief, bacterial strains were incubated in NB medium at 28°C with shaking at 175 rpm until an OD600 of 1.0 (109 CFU/ml) was attained. Rice leaves (6 weeks old for X. oryzae pv. oryzae and 8 weeks old for X. oryzae pv. oryzicola) were inoculated by cutting the leaves with sterile scissors dipped in bacterial suspension or by piercing the leaves with sterile needles dipped in bacterial suspension. Five plants and three leaves per plant were inoculated for each strain. Lesion lengths were measured 10 days after inoculation. This experiment was performed two times.

Supplementary Material

Supplemental material
supp_84_4_e02145-17__index.html (1,014B, html)

ACKNOWLEDGMENTS

This research was supported by the Special Fund for Agro-scientific Research in the Public Interest (201303023) and a grant (no. 2013-6) from the Innovation Team Program for Jiangsu Universities.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02145-17.

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