Additional file 1 of Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy
Creators: name: Dutta, Tushar K.
name: Vashisth, Neeraj
name: Ray, Soham
name: Phani, Victor
name: Chinnusamy, Viswanathan
name: Sirohi, Anil
Publisher: name: figshare
Year: 2023
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: Plant Biology

description: Additional file 1: Figure S1. Amino acid sequence alignment of A. thaliana HIPP27 protein (NCBI accession number: NP_201412) with HIPP27 proteins from Gossypium hirsutum (XP_040937216), G. arboreum (XP_017645826) and G. raimondii (XP_012472579). * and : indicate identical and similar amino acids, respectively. Figure S2. RT-qPCR-based expression analysis of hipp27 gene in different plant parts and developmental stages of A. thaliana Col-0. Fold change in expression was set as 1 in root tissue and 7 days-old-plant, and statistically compared with hipp27 expression in other plant parts and developmental stages, respectively (no significant difference was observed; Tukey’s HSD test, P &gt; 0.05). Gene expression was normalized using two housekeeping genes of A. thaliana (ubiquitin and 18S rRNA). Each bar represents the mean fold change value ± standard error (SE) of qPCR runs in three biological and technical replicates. Figure S3. RT-qPCR-based expression analysis of hipp27 gene and its homologues (hipp20, hipp21, hipp22, hipp23, hipp24, hipp25, hipp26) in genome edited line Athipp27-cr-12 and wild-type plants. Fold change in expression of target gene was set as 1 in wild-type, and statistically compared with expression in edited plants. Asterisk indicate significant difference (Tukey’s HSD test, P &lt; 0.01) in edited plant compared to the wild-type. Gene expression was normalized using two housekeeping genes of A. thaliana (ubiquitin and 18S rRNA). Each bar represents the mean fold change value ± standard error (SE) of qPCR runs in three biological and technical replicates. Figure S4. Comparative phenotyping of A. thaliana wild-type and HIPP27 mutant line for different growth parameters. Bars represent mean ± SE. Data were analyzed via Tukey’s HSD test, P &lt; 0.05. Bottom panel represents the images of 30-days-old plants in pots containing soil rite. Figure S5. RT-qPCR-based expression analysis of defense response genes in shoots (A) and roots (B) of M. incognita-infected A. thaliana wild-type and HIPP mutants at 2 days after inoculation. Fold change in expression was set as 1 in wild-types and statistically compared with expression in HIPP mutants (no significant difference was observed; Tukey’s HSD test, P &gt; 0.05). Gene expression was normalized using two housekeeping genes of A. thaliana (ubiquitin and 18S rRNA). Each bar represents the mean fold change value ± standard error (SE) of qPCR runs in three biological and technical replicates. Figure S6. Generation of transgenic Arabidopsis plants. (A) A. thaliana Col-0 plants at flowering stage suitable for transformation, (B) Plant transformation with Agrobactrium culture via floral dip method, (C) Exposure of plants to low temperature for vernalization, (D) Plants at pod stage for seed collection, (E) Dormancy breaking of T0 seeds, (F) seed germination in MS media supplemented with hygromycin antibiotic, (G) Screening of antibiotic resistant seedlings (red circles indicate seeds that did not germinate), (H) Transfer of 14-days-old seedlings (3-4 leaf stage) to pots containing soil rite, (I) Flowering initiated in 30-days-old plants, (J) Plants ready for genotyping and phenotyping analysis. Figure S7. Schematic representation of CRISPR/Cas9 vector construction. Amplification with the plasmid pCBC as a template was used to generate a PCR product containing the two targets (flanked by Bsa I endonuclease sites), gRNA scaffold, Arabidopsis U6 gene terminator (U6-26t) and promoter (U6-29p). PCR forward (F) and reverse (R) primers contained the two target sequences and Bsa I site. Two gRNA expression cassettes were assembled into the T-DNA portion of Cas9-expressing binary vector pHEE401 via Golden Gate cloning by replacing the spectinomycin resistance (SpecR) gene. In recombinant pHEE401 (pHEE401:Athipp27-cr), first gRNA expression cassette is driven by the Arabidopsis U6 promoter U6-26p. Codon optimized Cas9 expression is driven by an egg cell-specific promoter (EC1p) and rbcS E9 terminator (rbcS-E9t). NLS, nuclear localization signal; 35Sp, CaMV35S promoter; HygR, Hygromycin resistance; PolyA, CaMV35S terminator; LB, left border; RB, right border. Bottom panel depicts the nucleotide sequence of two sgRNA expression cassette generated in the current study. Gel photograph indicates PCR amplification of expected 846 bp fragment from different colonies of Agrobacterium tumefaciens GV3101 harboring the pHEE401:Athipp27-cr construct. M, 100 bp DNA ladder. Figure S8. Full-length gel image used in Figs. 4C and 5B, C. No overexposure was used.