[논문]Development of PCR-Based Molecular Marker for Detection of Xanthomonas campestris pv. campestris Race 6, the Causative Agent of Black Rot of Brassicas

Afrin, Khandker Shazia; Rahim, Md Abdur; Rubel, Mehede Hassan; Park, Jong-In; Jung, Hee-Jeong; Kim, Hoy-Taek; Nou, Ill-Sup

doi:10.5423/ppj.oa.06.2020.0103

Development of PCR-Based Molecular Marker for Detection of Xanthomonas campestris pv. campestris Race 6, the Causative Agent of Black Rot of Brassicas 원문보기

The plant pathology journal, v.36 no.5, 2020년, pp.418 - 427

Afrin, Khandker Shazia (Department of Horticulture, Sunchon National University) , Rahim, Md Abdur (Department of Horticulture, Sunchon National University) , Rubel, Mehede Hassan (Department of Horticulture, Sunchon National University) , Park, Jong-In (Department of Horticulture, Sunchon National University) , Jung, Hee-Jeong (Department of Horticulture, Sunchon National University) , Kim, Hoy-Taek (Department of Horticulture, Sunchon National University) , Nou, Ill-Sup (Department of Horticulture, Sunchon National University)

Abstract ▼ AI-Helper

Xanthomonas campestris pv. campestris (Xcc), the pathogen of black rot which is the most destructive disease of Brassica vegetables throughout the world. Here, we reported two novel sequence-characterized amplified region (SCAR) markers (i.e., XccR6-60 and XccR6-67) for the detection of Xcc race 6 via re-alignment of the complete genome sequences of Xcc races/strains/pathovars. The specificity of SCAR primer sets was verified by mean of PCR amplification using the genomic DNA template of Xcc races/strains/pathovars and two other plant infecting bacterial strains. The PCR result revealed that the XccR6-60 and XccR6-67 primer sets amplified 692-bp and 917-bp DNA fragments, respectively, specifically from race 6, while no visible amplification was detected in other samples. In addition, the SCAR primers were highly sensitive and can detect from a very low concentration of genomic DNA of Xcc race 6. However, the complete genome sequence of Xcc race 6 is not yet publicly available. Therefore, the cloning and sequencing of XccR6-60 and XccR6-67 fragments from race 6 provide more evidence of the specificity of these markers. These results indicated that the newly developed SCAR markers can successfully, effectively and rapidly detect Xcc race 6 from other Xcc races/strains/pathovars as well as other plant pathogenic bacteria. This is the first report for race-specific molecular markers for Xcc race 6.

주제어

표/그림 (8)

표 Table 1. Publicly available complete genome sequences of Xcc races, Xcc strains, and Xc subspecies, and their accession numbers
그림 Fig. 1. Whole genome alignment of the publicly available genome sequences of Xanthomonas campestris pv. campestris (Xcc) races, strains and pathovars. The syntenic blocks are connected among the Xcc races/strains/Xc pathovars. The variable region among the Xcc races/strains/Xc subspecies are shown with the line diagram in the bottom of the alignment. Genomic blocks are shown as colored rectangles.
표 Table 2. List of Xcc race 6 specific primers used for in this study
그림 Fig. 2. Gel electrophoresis (1.5% agarose) of the PCR products of Xanthomonas campestris pv. campestris (Xcc) race 6 specific sequence-characterized amplified region primer sets (A, XccR6-60, 692-bp; B, XccR6-67, 917-bp) amplified from genomic DNA of Xcc races and pathovars. Lane M, 100 bp plus DNA ladder; lanes 1-8, Xcc race 1-8; lane Xci, X. campestris pv. incane (Xci); lane Xcr, X. campestris pv. raphanin (Xcr); lane Xcv, X. campestris pv. vesicatoria (Xcv); lane Xag, X. axonopodis pv. glycines (Xag); lane Xad, X. axonopodis pv. dieffenbachiae (Xad); lane Xcz, X. campestris pv. zinniae (Xcz); lane Psm, Pseudomonas syringae pv. maculicola (Psm); lane Pcc, Pectobacterium carotovora subsp. carotovora (Pcc).
그림 Fig. 3. Sensitivity of the Xanthomonas campestris pv. campestris (Xcc) race 6 specific SCAR primers (XccR6-60 and XccR6-67). (A) XccR6-60. (B) XccR6-67. Lane M, 100 bp plus DNA ladder; lane 1, 50 ng/μl gDNA; lane 2, 5 ng/μl; lane 3, 0.5 ng/μl; lane 4, 0.05 ng/ μl; lane 5, 0.005 ng/μl; lane 6, negative control. For the PCR amplification, 1 μl DNA of Xcc race 5 from each dilutions was used as a template.
그림 Fig. 4. Nucleotide and amino acid sequences of the XccR6-60 (A) and XccR6-67 (B) genomic fragments cloned and sequenced from Xanthomonas campestris pv. campestris race 6. The open reading frames are presented in grey highlighted area: XccR6-60, 381-bp and XccR6-67, 720-bp. The deduced amino acid sequences are shown in the above of the nucleotide sequences: XccR6-60, 126-aa and XccR6-67, 239-aa. The nucleotide sequence is shown as reverse complement.
그림 Fig. 5. Detection of Xanthomonas campestris pv. campestris (Xcc) race 6 by bio-PCR with artificially Xcc (races 1-8, R1-R8) inoculated cabbage leaves with XccR6-60 (A) and XccR6-67 (B) primer sets. Lanes 1-24, artificially inoculated leaf samples with Xcc races (lanes: 1-3, race 1; 4-6, race 2; 7-9, race 3; 10-12, race 4; 13-15, race 5; 16-18, race 6; 19-21, race 7; 22-24, race 8; +ve, gDNA of Xcc race 5 as a positive control; –ve, negative control; M, 100 bp plus DNA ladder).
표 Table 3. The results of NCBI BLAST search with the Xcc race 6 specific genomic fragments

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, 2017). In this study, we aimed to develop molecular markers specific to Xcc race 6.

제안 방법

1) and absent in the remaining genomes in the alignment. In the next step, we designed two sets of primers on XccR6-60 (693-bp) and XccR6-67 (923-bp) genomic fragments considering CN14, CN15, and CN16 as reference genome and amplified by the PCR.
In this study, we developed PCR-based SCAR markers (XccR6-60 and XccR6-67) specific to Xcc race 6 through re-alignment of available complete genome sequences of fifteen Xcc races/strains/pathovars (Fig. 1). As the complete genome sequence of Xcc race 6 is not available yet, the variable genomic regions of known Xcc genome sequences were considered as targets to develop the race 6-specific molecular markers.
The availability of the complete genome sequences of Xcc races, Xcc strains, Xc pathovars and their re-alignment enabled us to develop Xcc race specific molecular markers. In this study, we developed two SCAR markers XccR6-60 and XccR6-67, for rapid detection of race 6 from other Xcc races (Fig. 1). Since, the complete genome sequence of Xcc race 6 is not available yet, we analyzed the variable genomic regions among the aligned genomes.
(2019) to develop Xcc race 5-specific molecular marker whose complete genome is not yet sequenced. The SCAR markers developed in this study were able to detect and identify Xcc race 6 from other Xcc races/strains/pathovars (Fig. 2, Supplementary Fig. 1). Further, these markers can detect Xcc race 6 from very low concentration of race 6 gDNA templates (total 0.

대상 데이터

Subsequently, the plasmid DNA was isolated and purified with a GeneAll kit (Seoul, Korea). The plasmid DNA samples were sequenced by ABI 3730XL DNA sequencer (Macrogen Inc., Seoul, Korea) using universal primers. The sequence alignment was carried out with the Clustal Omega online tool (https://www.

이론/모형

, Seoul, Korea) using universal primers. The sequence alignment was carried out with the Clustal Omega online tool (https://www.ebi.ac.uk/Tools/ msa/clustalo/).

성능/효과

2). Besides, the NCBI BLAST search of the cloned sequence of XccR6-60 and XccR6-67 fragments from Xcc race 6 showed more than 99% and 97%, respectively identity with three race-unknown Xcc strains, CN14, CN15, and CN16 (Table 3). Therefore, it can be anticipated that these race-unknown Xcc strain (CN14, CN15, and CN16) might belong to race 6.
In conclusion, the newly developed SCAR markers (XccR6-60 and XccR6-67) could be useful for rapid, lowcost and reliable detection of race 6 from other Xcc races/ strains/pathovars. Thus, these molecular markers would be valuable for early detection of this pathogen from infected Brassica crops worldwide.
1). Moreover, the sensitivity test of the SCAR primer sets revealed that they were sensitive enough to detect race 6 by PCR from total 0.05-0.5 ng template gDNA (Fig. 3).
The cloning and sequence analysis of XccR6-60 and XccR6-67 marker regions revealed that they have a considerably high homology with DUF4189 domain-containing protein (WP_076054737.1) and hypothetical protein (WP_076054735.1) of X. campestris, respectively. Further, domain analysis using protein sequence of XccR6-67 (239- aa) revealed that presence of TrbL domain (TrbL/VirB6 plasmid conjugal transfer protein, pfam04610) (Supplementary Fig.
Thereafter, BLASTp search was performed using these proteins in the NCBI. The result showed that 129-aa protein sequence of XccR6-60 had a significant homology (99% identity) with DUF4189 domain-containing protein of X. campestris (WP_076054737.1). In case of XccR6-67, 239-aa protein sequence had a significant homology with a hypothetical protein of X.
Since, the complete genome sequence of Xcc race 6 is not available yet, we analyzed the variable genomic regions among the aligned genomes. The result showed that the XccR6-60 and XccR6-67 fragments were specific to three race-unknown Xcc strains, including CN14, CN15, and CN16 (Fig. 1) and absent in the remaining genomes in the alignment. In the next step, we designed two sets of primers on XccR6-60 (693-bp) and XccR6-67 (923-bp) genomic fragments considering CN14, CN15, and CN16 as reference genome and amplified by the PCR.
gov/orffinder/). The results indicated that the XccR6-60 and XccR6-67 fragments contained 381-bp (129-aa) and 720-bp (239- aa) ORF, respectively (Fig. 4, Supplementary Figs. 3 and 4). Thereafter, BLASTp search was performed using these proteins in the NCBI.

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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