Journal: Scientific Reports

Article Title: Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci ( ClaPMR2 ) during watermelon- Podosphaera xanthii interaction using RNA-Seq and whole-genome resequencing approach

doi: 10.1038/s41598-020-70932-z

Figure Lengend Snippet: Processed RNA-seq data. ( A ) Total number of clean, mapped reads (watermelon) and bases of each mRNAseq sample (three replicates) following PM inoculation collected from watermelon lines: USVL677-PMS & USVL531-MDR at 0 h, 24 h, 72 h and 8 dpi. ( B ) Distribution of differentially expressed genes (DEGs) for each mRNAseq sample following PM inoculation collected from watermelon lines: USVL677-PMS & USVL531-MDR at 0 h, 24 h, 72 h and 8 dpi. The DEGs are shown in red logFC > 1, P value < 0.05 and FDR ratio of < 0.05 for each gene in each pair-wise comparison of each time points with uninoculated 0 h as control. The red dots highlight transcripts of positive and negative values of log2 Fold Change (logFC), indicating that the sequences were upregulated and downregulated at each time point. The black dots indicate non-differentially expressed genes. The genes names, fold changes, and p values, FDR values for up- and down-regulated DEGs were listed in Table . ( C ) Table showing total DEGs, upregulated and downregulated genes in response to PM in USVL677-PMS & USVL531-MDR at 24 h, 72 h and 8 dpi. ( D ) Multidimensional scaling (MDS) of all replicate samples.

Article Snippet: For whole-genome resequencing, high-quality genomic DNA was extracted from leaves of four week old watermelon seedlings (USVL531-MDR, USVL677-PMS, and four RILs: R-201, R-202, R-203 and R-204, having 1–2 fully expanded true leaves using the Qiagen DNeasy Plant Mini Kit as suggested by the manufacturer’s instructions (Qiagen, Hilden, Germany).

Techniques: RNA Sequencing, Comparison, Control

Journal: Scientific Reports

Article Title: Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci ( ClaPMR2 ) during watermelon- Podosphaera xanthii interaction using RNA-Seq and whole-genome resequencing approach

doi: 10.1038/s41598-020-70932-z

Figure Lengend Snippet: Time course PM infection process in USVL531-MDR and USVL677-PMS watermelon lines . ( A ) PM conidial development on leaves of 3–4 week old watermelon plant USVL531-MDR: Powdery mildew resistant line; USVL677-PMS: powdery mildew susceptible line at 0 h, 24 h, 72 h and 8 dpi with powdery mildew fungus; ( B ) Trypan-blue stained hyphae collected from watermelon lines: USVL677-PMS & USVL531-MDR at 0 h, 24 h, 72 h and 8 dpi with powdery mildew fungus; ( C ) Bar graph representing PM conidial count on leaves of USVL677-PMS, USVL531-MDR at 8 dpi. Asterisks indicate data statistically significant from that of susceptible line (USVL677-PMS; P < 0.05). ( D ) Bar graph representing P. xanthii secretome transcript counts on leaves of USVL677-PMS, USVL531-MDR at 0 h, 24 h, 72 h and 8 dpi. Asterisks indicate data statistically significant from that of susceptible line (USVL677-PMS; P < 0.05).

Article Snippet: For whole-genome resequencing, high-quality genomic DNA was extracted from leaves of four week old watermelon seedlings (USVL531-MDR, USVL677-PMS, and four RILs: R-201, R-202, R-203 and R-204, having 1–2 fully expanded true leaves using the Qiagen DNeasy Plant Mini Kit as suggested by the manufacturer’s instructions (Qiagen, Hilden, Germany).

Techniques: Infection, Staining

Journal: Scientific Reports

Article Title: Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci ( ClaPMR2 ) during watermelon- Podosphaera xanthii interaction using RNA-Seq and whole-genome resequencing approach

doi: 10.1038/s41598-020-70932-z

Figure Lengend Snippet: Summary of whole-genome re-sequencing (WGR) data of multiple disease resistant watermelon line: USVL531-PMR; PM susceptible watermelon line: USVL677-PMS; introgressed RILs: R-201, R-202, R-203, R-204.

Article Snippet: For whole-genome resequencing, high-quality genomic DNA was extracted from leaves of four week old watermelon seedlings (USVL531-MDR, USVL677-PMS, and four RILs: R-201, R-202, R-203 and R-204, having 1–2 fully expanded true leaves using the Qiagen DNeasy Plant Mini Kit as suggested by the manufacturer’s instructions (Qiagen, Hilden, Germany).

Techniques:

Journal: Scientific Reports

Article Title: Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci ( ClaPMR2 ) during watermelon- Podosphaera xanthii interaction using RNA-Seq and whole-genome resequencing approach

doi: 10.1038/s41598-020-70932-z

Figure Lengend Snippet: Result summary of comparative SNP analysis among USVL531-PMR, USVL677-PMS, R-201, R-202, R-203, R-204 and the watermelon reference genome 97103.

Article Snippet: For whole-genome resequencing, high-quality genomic DNA was extracted from leaves of four week old watermelon seedlings (USVL531-MDR, USVL677-PMS, and four RILs: R-201, R-202, R-203 and R-204, having 1–2 fully expanded true leaves using the Qiagen DNeasy Plant Mini Kit as suggested by the manufacturer’s instructions (Qiagen, Hilden, Germany).

Techniques:

Journal: Scientific Reports

Article Title: Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci ( ClaPMR2 ) during watermelon- Podosphaera xanthii interaction using RNA-Seq and whole-genome resequencing approach

doi: 10.1038/s41598-020-70932-z

Figure Lengend Snippet: Result summary of comparative insertions and deletions (INDELS) analysis among USVL531-PMR, USVL677-PMS, R-201, R-202, R-203, R-204 and the watermelon reference genome 97103.

Article Snippet: For whole-genome resequencing, high-quality genomic DNA was extracted from leaves of four week old watermelon seedlings (USVL531-MDR, USVL677-PMS, and four RILs: R-201, R-202, R-203 and R-204, having 1–2 fully expanded true leaves using the Qiagen DNeasy Plant Mini Kit as suggested by the manufacturer’s instructions (Qiagen, Hilden, Germany).

Techniques:

Journal: Scientific Reports

Article Title: Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci ( ClaPMR2 ) during watermelon- Podosphaera xanthii interaction using RNA-Seq and whole-genome resequencing approach

doi: 10.1038/s41598-020-70932-z

Figure Lengend Snippet: Circos plots showing the distribution of SNPs, InDels, NBS-LRR and Mlo genes in PM resistance introgressed lines. The outer ring represents the eleven watermelon chromosomes. The scatter plot inside this ring shows the distribution of homozygous SNPs (dark green) and InDels (red) between ( A ): Citrullus lanatus subsp. Vulgaris cv. 97103 and R-201; ( B ): Citrullus lanatus subsp. Vulgaris cv. 97103 and R-202; ( C ): Citrullus lanatus subsp. Vulgaris cv. 97103 and R-203; ( D ): Citrullus lanatus subsp. Vulgaris cv. 97103 and R-204. The green line represents the genotype percentage of SNPs and orange line indicates the genotype percentage of InDels in CSK lines from the donor parent USVL531-MDR along the chromosomes. The NBS-LRR and Mlo gene locations are represented in orange and blue lines respectively along the chromosomes. Black dotted rectangle area represents the chrs 2 region with increased genotype percentage of SNPs and InDels with 7 NBS-LRR and one Mlo gene. Circos-0.69-6 software was used to generate the circos plot ( https://circos.ca/software/download/circos/) .

Article Snippet: For whole-genome resequencing, high-quality genomic DNA was extracted from leaves of four week old watermelon seedlings (USVL531-MDR, USVL677-PMS, and four RILs: R-201, R-202, R-203 and R-204, having 1–2 fully expanded true leaves using the Qiagen DNeasy Plant Mini Kit as suggested by the manufacturer’s instructions (Qiagen, Hilden, Germany).

Techniques: Software

Journal: Scientific Reports

Article Title: Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci ( ClaPMR2 ) during watermelon- Podosphaera xanthii interaction using RNA-Seq and whole-genome resequencing approach

doi: 10.1038/s41598-020-70932-z

Figure Lengend Snippet: Predicted Secondary Structure of watermelon ClaPMR2 . ( A ) Open reading frame of ClaPMR2 protein with predicted secondary structure and transmembrane domains using EMBOSS 6.5.7 (Geneious Prime v2019.2.1, https://www.geneious.com/prime/ ). The ORF consists of the conserved for the N terminal RPW8-EHM targeted motif. The Predicted secondary structure of alpha helix, beta strand, coil and turn are presented in purple cylinders, yellow arrows, grey sinusoids and blue curved arrow. Detailed information of individual sequences is presented in Table . ( B ) Comparative secondary structure and transmembrane domains analysis of Arabidopsis RPW8.1, RPW8.2 with the watermelon ClaPMR2 using EMBOSS 6.5.7 (Geneious Prime v2019.2.1, https://www.geneious.com/prime/ ). ( C ) Comparative percentage of sequence (aa) similarity of ClaPMR2 / Cla019831 with AtRPW8.1 and AtRPW8.2 domain. (D) 3D modeling with structural overlap of the RPW8 domain of Arabidopsis thaliana (AtRPW8.1) with the N-terminal domain of watermelon, Cla-RPW8 (USVL531-MDR). Geneious Prime v2019.2.1, https://www.geneious.com/prime/ ) was used to generate the 3D structure model. ClaPMR2 expression level at 8dpi in USVL531-MDR and USVL677-PMS.

Article Snippet: For whole-genome resequencing, high-quality genomic DNA was extracted from leaves of four week old watermelon seedlings (USVL531-MDR, USVL677-PMS, and four RILs: R-201, R-202, R-203 and R-204, having 1–2 fully expanded true leaves using the Qiagen DNeasy Plant Mini Kit as suggested by the manufacturer’s instructions (Qiagen, Hilden, Germany).

Techniques: Sequencing, Expressing

Journal: Scientific Reports

Article Title: Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci ( ClaPMR2 ) during watermelon- Podosphaera xanthii interaction using RNA-Seq and whole-genome resequencing approach

doi: 10.1038/s41598-020-70932-z

Figure Lengend Snippet: SNPs derived CAPS validation on Parent, F 2 and RIL introgressed watermelon lines using DNA Agarose gel. ( A ) Comparative nucleotide and amino acid consensus sequences with predicted secondary structure alignment of ClaPMR2 encoding protein in Parent (USVL531-MDR & USVL677-PMS), and RIL introgressed watermelon lines (R-201, R-202, R-203, R-204). Black line rectangle area HRH (C-T; Arg-STOP), represents the location of SNP region with substitution position Arg-STOP codon in ( ClaPMR2, Citrullus lanatus PM Resistance gene ). ( B ) DNA gel electrophoresis showing the CAPS marker analysis on ClaPMR2 gene in parent lines: USVL531-MDR & USVL677-PMS; RILs: R-201, R-202, R-203 and R-204 and F 2 populations. Since the resistant phenotype is dominant, we observed ClaPMR2 loci , cosegregated with the resistant locus in heterozygous and homozygous individuals with PM resistance lines.

Article Snippet: For whole-genome resequencing, high-quality genomic DNA was extracted from leaves of four week old watermelon seedlings (USVL531-MDR, USVL677-PMS, and four RILs: R-201, R-202, R-203 and R-204, having 1–2 fully expanded true leaves using the Qiagen DNeasy Plant Mini Kit as suggested by the manufacturer’s instructions (Qiagen, Hilden, Germany).

Techniques: Derivative Assay, Biomarker Discovery, Agarose Gel Electrophoresis, DNA Gel Electrophoresis, Marker

Journal: Scientific Reports

Article Title: Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci ( ClaPMR2 ) during watermelon- Podosphaera xanthii interaction using RNA-Seq and whole-genome resequencing approach

doi: 10.1038/s41598-020-70932-z

Figure Lengend Snippet: Proposed model of ClaPMR2 mediated resistance signaling in watermelon in response to P. xanthii . During the infection process, the plant pathogenic fungi, P. xanthii develop highly specialized structures known as haustoria to secrete specific effector molecules. These effectors are recognized by the sophisticated plant specific intracellular immune receptors (NBS-LRRs, ClaPMR2 ) to activate the ETI mediated plant defense response by activating shikimate pathway to produce elevated levels of melatonin (MEL) and salicylic acid (SA) in chloroplast. The increased SA levels further potentiate downstream signaling by triggering nuclear gene expressions of various pathogenesis-related (PR1, PR5) and other defense related genes providing resistance to pathogen. During host–pathogen interaction the pathogenic fungi can alters expression of diverse defense related genes from initial germination of conidia to hyphal development and proliferation during the 8-day time period activating diverse intra- and extra-cellular resistance proteins downstream to ClaPMR2 MEL (M): melatonin; ETI: effector triggered immunity; TFs: transcription factors; MAPKKK: map kinase kinase kinase; MAPKK: map kinase kinase; MAPKKK: map kinase; NBS-LRR: nucleotide-binding site leucine-rich repeat.

Article Snippet: For whole-genome resequencing, high-quality genomic DNA was extracted from leaves of four week old watermelon seedlings (USVL531-MDR, USVL677-PMS, and four RILs: R-201, R-202, R-203 and R-204, having 1–2 fully expanded true leaves using the Qiagen DNeasy Plant Mini Kit as suggested by the manufacturer’s instructions (Qiagen, Hilden, Germany).

Techniques: Infection, Expressing, Binding Assay