Editseq

To exclude genes known to be the cause of colistin resistance in K. pneumoniae from complementation analysis, KPN_02065, KPN_02066, KPN-02067, and KP1_3468 genes were matched against the crrAB region and mgrB gene [5 (link),28 (link)]. The crrAB region and mgrB sequence database was provided by the National Center for Biotechnology Information (NCBI). A comparison of the orthologous sequence was analyzed by DNAstar EditSeq and MegAline (DNASTAR, Inc., Madison, WI, USA).

Bisulfite-converted DNA from control and infected bAM was amplified in 25 µl reactions containing 0.2 µM primers, 1× buffer, 0.2 mM dNTPs, 2.5 U Platinum Taq DNA polymerase and 3 mM MgCl₂. Primer sequences are detailed in Table 1. PCR cycling conditions were as follows: 95 °C for 3 min followed by 35 cycles of 30 sec each at 95 °C; 58 °C, 72 °C for 30 sec, and a final elongation step of 5 min at 72 °C. PCR products were purified using the Wizard clean up kit (Promega) and cloned into the pJET1.2/blunt vector (Fermentas). Insertion of PCR products was verified by digestion with BglII and positive clones were sequenced using conventional Sanger sequencing (Eurofins Genomics). Combined bisulfite restriction analysis (COBRA) was carried out using TaqαI, and/or AciI as outlined in^{27 (link)}. Sequence analysis and alignment was performed using DNAStar EditSeq, MegAlign (www.dnastar.com) and BiQ Meth Analyzer (http://biq-analyzer.bioinf.mpi-inf.mpg.de). During this analysis, sequences with low C-T conversion rate (<95%) and with a high number of sequencing errors (sequence identity with genomic sequence less than 80%) were excluded from the alignment. Identical clones were also excluded from the analysis.

The amino acid sequence of CL25983Contig1 was predicted using a DNAstar program Editseq option (DNASTAR, Inc., Madison, Wis.). A phylogenetic tree was constructed using the amino acid sequence deduced from CL25983Contig1 and 93 S-RNases from other species by a Neighbor-joining method by using MEGA 5.2 (www.megasoftware.net). Total RNA was extracted from 8 different tissues. The expression pattern of CL25983Contig1 was examined using qRT-PCR with a reference gene GAPDH. Furthermore, the expression profiling of CL25983Contig1 was detected in both two pollination treatments from 0 h to 120 h after pollination. Each reaction was performed with three biological replicates and three technical replicates.