Simple Deletion: a vector- and marker-free method to generate and isolate site-directed

Transcription

1 Electronic supplementary materials Simple Deletion: a vector- and marker-free method to generate and isolate site-directed deletion mutants Yasuhiro Inoue 1, Seiji Tsuge 2 1 National Agriculture and Food Research Organization, National Agricultural Research Center, Tsukuba, Ibaraki, , Japan 2 Graduate School of Agriculture, Kyoto Prefectural University, Kyoto, Japan Corresponding author: Yasuhiro Inoue 1

2 Fig. S1 PCR amplification test to generate Xanthomonas campestris pv. campestris mutants with a kanamycin-resistance gene instead of the 20.4-kb hrp gene cluster. Kanamycin-resistant clones and the parental strain were used as templates for PCR analyses with primers 7 and 8 (a), 9 and 10 (b), and 5 and 6 (c). Lanes 1 10, kanamycin-resistant clones; M, 1 kb Plus DNA ladder (Invitrogen, Carlsbad, CA, USA); P, parental strain. In clones containing an ectopic mutation, a 24.4-kb fragment derived from the wild-type hrp region was expected to be amplified by PCR using primers 5 and 6, but it was not produced under the condition tested (c, lanes 1 7) 2

3 Fig. S2 Effect of DNase I on elimination of the inoculum DNA in culture. One microgram pdhrp was added to 1 ml of Xanthomonas campestris pv. campestris culture with (lanes 4 6) or without (lanes 1 3) 35 units DNase I. One microlitre of the culture was used for PCR with primers 7 and 8. The arrow indicates the 574-bp fragment derived from the inoculum DNA 3

4 Generating of deletion mutants by using the Simple Deletion method Construction of DNA fragments for homologous recombination The DNA fragments used in the homologous recombination experiment to obtain hrpg and hrpg-hrpx deletion mutants of Xanthomonas campestris pv. campestris and an hrpf-hrpc deletion mutant of Ralstonia solanacearum were constructed as follows. Xanthomonas campestris pv. campestris (Xcc) strain H7601 (MAFF ) and Ralstonia solanacearum (Rs) strain 8242 (MAFF107633) were used. Primers were arranged in a similar manner to those used for the Xcc deletion mutant of the hrp genes cluster (Fig. 1). Primer sequences are listed in Table S1. Bacterial suspensions (5 µl) were heated for 2 min at 98 C and used as templates for PCR amplification. Because of the high GC content of the sequences flanking the hrpg-hrpx region of Xcc and the hrpf-hrpc region of Rs, we tried various kinds of DNA polymerases (as described below) to achieve optimal amplification of each DNA fragment from each template. In the case of Xcc mutants, the upstream and the downstream regions were both amplified using LA Taq DNA polymerase with 2 GC buffer I (Takara Bio, Otsu, Japan) and the reaction solution recommended by the manufacturer. The PCR conditions were as follows: 2 min at 95 C; 30 cycles of 30 s at 95 C, 30 s at 60 C, and 90 s at 72 C; and a final incubation for 7 min at 72 C. Amplified fragments were annealed with each other and then subjected to DNA extension and PCR amplification by using iproof High-Fidelity DNA polymerase (Bio-Rad, Hercules, CA) with primers 1 and 4, according to the manufacturer s instructions under the following conditions: 30 s at 98 C; 30 cycles of 15 s at 98 C, 15 s at 62 C, and 105 s at 72 C; and a final incubation for 5 min at 72 C. 4

5 For PCR amplification from Rs, KAPA2G Robust HotStart DNA Polymerase (KAPA Biosystems, Woburn, MA) was used according to the manufacturer s instructions under the following conditions: 3 min at 95 C; 30 cycles of 30 s at 95 C, 30 s at 57.5 C, and 90 s at 72 C; and a final incubation for 5 min at 72 C. Fragments were annealed with each other and then subjected to DNA extension and PCR by using Pyrobest DNA polymerase (Takara Bio) with primers 1 and 4, according to the manufacturer s instructions under the following conditions: 2 min at 95 C; 30 cycles of 30 s at 95 C, 30 s at 57.5 C, and 2 min at 72 C; and a final incubation for 5 min at 72 C. (Note: In these cases, primers 5 and 2 and primers 3 and 6 were used to amplify the region flanking the 5 end of the target (upstream region) and the region flanking the 3 end of the target (downstream region), respectively. When the fragments were connected with each other, a pfu-based enzyme was used with primers 1 and 4. These procedures decreased nonspecific amplification and made it possible to omit the T4 DNA polymerase treatment.) PCR conditions for sib selection to detect and isolate deletion mutants The nested PCRs using KAPA2G Robust HotStart DNA Polymerase (KAPA Biosystems) with GC buffer were performed according to the manufacturer s instructions. The primary PCR was performed under the following conditions: 3 min at 95 C; 30 cycles of 30 s at 95 C, 30 s at 57.5 C; and 33 s (for detection of the Xcc hrpg deletion mutant) or 50 s at 72 C (for detection of the Xcc hrpg-hrpx deletion mutant and Rs hrpf-hrpc deletion mutant); and a final incubation for 5 min at 72 C. The secondary PCR was performed under the following conditions: 3 min at 95 C; 30 cycles of 30 s at 95 C, 30 s 5

6 at 57.5 C; and a final incubation for 5 min at 72 C. The numbers of mutant-containing cultures detected at each step are listed in Table S2. Generation and isolation of the mutant was confirmed by performing PCR analyses (Fig. S3). 6

7 Table S1. Primers used for deletion of the hrpg gene and the hrpg-hrpx region of Xanthomonas campestris pv. campestris and the hrpf-hrpc region of Ralstonia solanacearum For 685-bp deletion of hrpg gene of Xcc Primer Position on AE a Sequence CACTGGCCGAGAAGACGA , TCGTACTGACGCACCTTCCAAGGGCATGAC , GCCCTTGGAAGGTGCGTCAGTACGAATGCC AAGCCGAAGATGTCTCCA TTGCGATGGGTGAGAAGG TTGGCCTGGCAATACTCG CGCCGTTGTCGTTTATGGG GGTCTACACCTGCACGTTG GCGCAGCTTGTAGATGTG ACTTGCTGGCCTGGTATC For 2963-bp deletion of hrpg-hrpx region of Xcc Position on AE a CACTGGCCGAGAAGACGA , TCCATCGGCGCTACCTTCCAAGGGCATGAC , GCCCTTGGAAGGTAGCGCCGATGGAAACCCT GAGCAACATGCCACGCAA TTGCGATGGGTGAGAAGG TTCGGAGAGTGCGTGTTG CGCCGTTGTCGTTTATGGG TGCGAGCCGGAAAGGATAG GCGCAGCTTGTAGATGTG ACTTGCTGGCCTGGTATC For 2545-bp deletion of hrpf-hrpc region of Rs Position on AL b ATGCCGGATCAGGCAGACAC , CTGGTATCGAACCAGGCATCGTTGGCGTCCTTGATGAG , CTCATCAAGGACGCCAACGATGCCTGGTTCGATACCAG CGCAACCGCGTTCATATCGG TCATGATCCAGGGTCCGAC GCGTCCTGCATGTAGGTC CGGTTCCCTCGATCTATCC GGAGACGTTGTCGATGAGG AGGACGAAGACGCCGTATC GTGCGATCTCGTCAAGCAG a GenBank accession number of the genome sequence of Xcc ATCC b GenBank accession number of the mega plasmid sequence of Rs GMI

8 Table S2. PCR-based sib selection to isolate hrpg and hrpg-hrpx deletion mutants of Xanthomonas campestris pv. campestris and a hrpf-hrpc deletion mutant of Ralstonia solanacearum Sib selection step 685 bp of hrpg gene of Xcc 2963 bp of hrpg-hrpx region of Xcc 2545 bp of hrpf-hrpc region of Rs 1 3/96 a) 6/96 4/96 2 1/48 5/48 4/48 3 1/48 3/48 3/48 4 5/48 10/44 7/72 5 3/48 17/48 1/48 6 7/48 1/48 NT b) a) Numbers of samples containing mutants per numbers of samples tested are shown. b) After step 5, single colonies were isolated. Expected ratios of mutants to total bacteria in each sample are 1:10 6 (step 1), 1:10 5 (step 2), 1:10 4 (step 3), 1:10 3 (step 4), 1:10 2 (step 5) and 1:10 1 (step 6). 8

9 a b c Fig. S3 PCR amplification test for mutants. PCR analysis was used to detect Xanthomonas campestris pv. campestris (Xcc) mutants with a deletion of the hrpg gene (a), Xcc mutants with a deletion of the hrpg-hrpx genes (b), and Ralstonia solanacearum (Rs) mutants with a deletion of the hrpf-hrpc genes (c). Two mutants and the parental strain were used as templates for each PCR using primers 5 and 6 (lanes 1 3), 7 and 8 (lanes 4 6), or 9 and 10 (lanes 7 9). Lanes 1, 2, 4, 5, 7 and 8 correspond to the mutant strain; lanes 3, 6, 9 correspond to the parental strain; and M corresponds to the 1 kb Plus DNA ladder. Arrows mark expected fragments. In the parental strain of Xcc, a 6.23-kb fragment was expected to be amplified by PCR using primers 5 and 6, but it was not produced under our conditions (b; lane 3). Also in the parental strain of Rs, a 6.53-kb fragment was expected to be amplified by PCR using primers 5 and 6, but it was not produced under the conditions that we used (c; lane 3). 9