Primer design and quantitative reverse transcriptase qRT-PCR

The whole-genome sequence of calla lily is not available; thus, cDNA sequences of the monocot species Zea mays, Triticum aestivum, Oryza sativa, and of the dicot model plant Arabidopsis thaliana were aligned to identify the conserved sites across all species. Primers were designed, based on these conserved sites, and PCR was performed on the cDNA of the calla lily plant tissues (Table ​(Table1).1). PCR products were then sequenced in a 3730 DNA analyzer (Applied Biosystems, CA, USA) to obtain cDNA sequences of specific genes in calla lily (Zantedeschia sp.). These were further used to design primers for qRT-PCR (Table ​(Table1)1) to quantify the expression of defense-related genes in this study. PCR products of the latter primers were cloned into a pGEM-T vector using pGEM^®-T Easy Vector System kit (Promega, WI, USA). In all, 2 µl of PCR product was mixed with 50 µl of competent E. coli (TOP10) DH5α on ice, then a heat shock protocol was applied at 42 °C for 45 s followed by ice. The cells were incubated on a rotary shaker for 1 h at 37 °C and then plated on a selective medium containing 100 µg ml⁻¹ ampicillin, and additional 100 μl of isopropyl β-D-1-thiogalactopyranoside (IPTG) 0.1 M and 20 μl of 5-bromo-4-chloro-indolyl-β-D-galactopyranoside (X-GAL) 0.5 g ml⁻¹ for a blue-white screen. Transformed colonies were grown overnight with LB supplemented with 100 µg ml⁻¹ ampicillin and plasmid DNA was extracted with PureYield™ Plasmid Miniprep System (Promega, Madison, WI, USA) and sequenced for validation using the T7 primer 5’-TAATACGACTCACTATAGGG-3'⁴⁴.

The SYBR^® Green (Applied Biosystems, USA) qRT-PCR assay was used to determine the expression of defense-related genes in ZA and CR following challenge infection with Pb. Real-time PCR amplifications were performed in Step One Plus real-time PCR system (Applied Biosystems, CA, USA) using gene-specific primers (Table ​(Table1),1), as previously reported³⁵. The data were analyzed by the comparative C_T (ΔΔC_T) method, with expression normalized to the expression of the reference gene actin.