*4.6. Quantification of Plant Cell Death by Trypan Blue Staining and Ion Leakage Assay*

To visualize dead plant cells and to measure the cell death area of infected leaves, trypan blue staining was performed on the leaves of four-week old infected *A. philoxiroides* and *A. sessilis*. Leaves were removed at each time interval (0, 24, 48, 72, and 96 hpi) and stained with lactophenol-trypan blue, followed by de-staining with saturated chloral hydrate using the method of Koch and Slusarenko [108]. Samples were photographed every day before and after staining to measure the diameter of the infected area using ImageJ [109]. To further confirm cell death, ion leakage assay was performed on infected leaves of both species using an electrolytic conductivity meter (model P772) following the procedure outlined in Hatsugai and Katagiri [110].

## *4.7. Flanking Sequence Isolation and Bioinformatic Analyses*

Six isolated DNA sequences from both *A. philoxeroides* and *A. sessilis* (Table S3) were searched in the NCBI non-redundant protein database using BLAST+ (version 2.2.31) to compare with other plant species. BLASTx output of each sequence was submitted to OrfPredictor [111] to identify the best matching open reading frame (ORF) sequence [110] (Table S4). Furthermore, one gene from each of the hormones, *PAL* (SA), *JAR1* (JA), and *EIN3* (ET) was selected for additional flanking sequence isolation for both *A. philoxiroides* and *A. sessilis* (for detailed function and bioinformation analysis). Flanking (50 and 30 ) sequences were isolated from a known sequence region (i.e., initially isolated sequences, Table S2) using 50 Genome walking and 30 RACE (Rapid Amplification of cDNA Ends) techniques, following the methods outlined in the respective kit instructions (Genome Walking Kit, code 6108 and 3 0 -Full RACE Core Set with PrimeScript RTase, code 6106, Takara, Shiga, Japan). Isolated sequences from both techniques were assembled for each gene with the corresponding initial sequence using CAP3 sequence assembly program with the following parameters: base quality cut-off for clipping value of 12, overlap length cut-off value ≥20, overlap percent identity ≥75, and overlap similarity score ≥500 [112]. The assembled single long contig was selected for annotating protein coding gene with ab-initio method and *Beta vulgaris* gene-specific parameters using the FGENESH online tool [113]. Predicted CDS and peptide sequences were reconfirmed by aligning to the NCBI RefSeq nucleotide and protein database using mega BLAST and BLASTp search tools. Clustal-W from MEGA 7.0.26 was used for multiple sequence alignment of each peptide sequence with other related species. A maximum likelihood phylogenetic tree was constructed with 1000 bootstrap replicates (Figure S2) [114]. In addition, motifs were searched for each peptide sequences using MEME suite [115] with the default options. The conserved domain was searched using the NCBI database with an expected value of 0.010000 [116].
