Additional file 2 of TALome and phenotypic analysis of Pakistani Xanthomonas oryzae pv. oryzae population revealed novel virulent TALEs contributing to bacterial blight of rice

Additional file 2: Figure S1. Geographic distribution per site and per year of a collection of 101 strains of Xanthomonas oryzae pv. oryzae from Pakistan. A collection of 101 strains from two provinces (39 from Punjab and 62 from Khyber Pakhtunkhwa) of Pakistan were used in this study. All the strains were isolated from leaves collected in 2017 (n = 40), 2018 (n = 22), 2019 (n = 24), and 2021 (n = 15). Sampled districts are block shaded, and the number of strains plus collection year is denoted with pie charts. The map was generated via QGIS, representing provincial boundaries in bold black lines. Figure S2. PCR-based validation of Pakistani Xoo isolates. A total of 101 Pakistani Xoo strains were screened using a specific set of primers; Xoo80F/Xoo80R amplifies a 162 bp fragment. PXO99A was used as positive control, and PCR without template DNA as negative (-) control. All strains were isolated from the rice-producing areas in two provinces; Punjab and Khyber Pakhtunkhwa of Pakistan from 2017–2019 and 2021. Strain names are indicated at the top; Lane M represents marker size in base pairs (bp). Figure S3. Southern blotting of 101 Pakistani Xoo isolates. The genomic DNA was subjected to BamHI digestion, transferred onto nylon membranes, and subsequently probed with the SphI fragment from PthXo1. Hybridization results unveiled distinct genotypes, denoted as G1 to G11, corresponding to the respective strains indicated at the top. The strains PXO99A and LN18 were used for reference. λEcoT14 marker is shown in the left lane in base pairs (bp). Figure S4. Inoculation phenotypes of Pakistani Xoo genotypes on cv. IR24 and Kitaake NILs. a Disease phenotypes of NILs containing different R genes and, Kitaake and b its EBE defective rice lines after inoculation with Pakistani Xoo genotypes. These phenotypes depict the functional relationship between TAL effectors and their targeted resistance/susceptibility genes. PXO99A, PXO86, and LN18 were used as reference strains. Photos were taken 14 days post-inoculation. Representative disease lesions are shown in the figure and mean lesion lengths ± SD (n = 8) are provided in Additional file 1: Table S3. Rice lines and the respective strain names are mentioned at the top of each figure. Phenotypes of CBB23, IRBB5, and IRBB4 show resistance to 101, 27, and 1 strain, respectively. Whereas phenotypes of cv. Kitaake and its EBE defective rice lines revealed that the majority of Pakistani Xoo stains harbor PthXo1-like effector (78 strains), one strain carries PthXo3/AvrXa7 like effector, and 20 strains contain a variant of PthXo2 like effector (PthXo2**).