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, 9 (1), 2039

The Rice Blast Resistance Gene Ptr Encodes an Atypical Protein Required for Broad-Spectrum Disease Resistance

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The Rice Blast Resistance Gene Ptr Encodes an Atypical Protein Required for Broad-Spectrum Disease Resistance

Haijun Zhao et al. Nat Commun.

Abstract

Plant resistance genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. Here we show that Ptr is an atypical resistance gene encoding a protein with four Armadillo repeats. Ptr is required for broad-spectrum blast resistance mediated by the NLR R gene Pi-ta and by the associated R gene Pi-ta2. Ptr is expressed constitutively and encodes two isoforms that are mainly localized in the cytoplasm. A two base pair deletion within the Ptr coding region in the fast neutron-generated mutant line M2354 creates a truncated protein, resulting in susceptibility to M. oryzae. Targeted mutation of Ptr in a resistant cultivar using CRISPR/Cas9 leads to blast susceptibility, further confirming its resistance function. The cloning of Ptr may aid in the development of broad spectrum blast resistant rice.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Cloning and characterization of Ptr. a Ptr was mapped between SSR RM3246 and RM1047 on chromosome 12 (Chr. 12). b, c Fine mapping of Ptr to 63 kb. d Predicted open reading frames (ORFs) with indicated direction of transcription. e Diagram of Ptr, mutations identified in M2354 and Amane. Exons are indicated by black filled rectangles. Armadillo (ARM) repeats (Superfamily 1.75) are indicated by the red filled rectangles. Insertion/deletion (InDel) is indicated by triangles. f The protein sequence of Ptr in Katy. Black rectangle indicates 41 missing amino acids in A isoform (864 amino acids) compared to B isoform (905 amino acids). The ARM repeats are underlined and polymorphic regions are indicated by triangles. Compared to Katy, the 2 bp (GG) deletion of Ptr in M2354 resulted in a truncated protein with 645 amino acids, while five different amino acids, as well as a four amino acid (KPEK) insertion in Amane, are indicated in red. g Subcellular localization of the Ptr-green fluorescent protein (GFP) fusion proteins in rice protoplasts expressing cytoplasmic/nuclear-localized red fluorescent protein (RFP). Pictures were taken 16-h after protoplast transfection. GFP, RFP, differential interference contrast (DIC), and merged channels are indicated on the top. Scale bars represent 10 μm
Fig. 2
Fig. 2
CRISPR/Cas9-mediated mutation of Ptr in a resistant cultivar. a The Ptr gene was targeted with two specific gRNAs to achieve knockouts. CRISPR/Cas9 editing resulted in InDels of differing length or a fragment deletion between the two protospacer (PS) sites are indicated by asterisk (*). b Disease symptoms on wild-type Katy and CRISPR-edited Ptr mutant leaves after spray-inoculation with AVR-Pita-containing strains IB-49 (ML1) or IC-17 (ZN57). c Disease rating of Katy and Ptr mutant plants at 6 days post-inoculation with 0–5 rating scale where 0–2 is resistant and 3 to 5 is susceptible. d Average lesion length on Katy and Ptr mutant leaves at 6 days post-inoculation. Error bars indicate standard deviation (s.d.) of average lesion area and disease rating. Data represent means ± s.d. (n = 12)
Fig. 3
Fig. 3
Disease reactions of Pi-ta/Pi-ta2/Ptr differential lines. a Disease reactions of Pi-ta/Pi-ta2/Ptr lines to four diverse races/isolates. Disease reaction was evaluated 7 days post-inoculation with 0–5 rating scale where 0–2 is resistant and 3 to 5 is susceptible. Rice varieties with the Pi-ta/Pi-ta2/Ptr genes were susceptible to IE-1K (TM2 lacking AVR-Pita) and resistant or moderately resistant to the transformant IE-1K + AVR-Pita from O-137. Note: M. oryzae races (isolates), IB-1 (15A6_3, 15L22_3), IB-17 (SSID116), IB-49 (ML1), and IE-1 (14A15–2), differentiated the Pi-ta-containing rice lines from the rice lines with Pi-ta2/Ptr. b Photographs showing disease reaction of indicated rice lines and races: Katy (Pi-ta+/Pi-ta2+/Ptr+), Amane (Pi-ta+/Pi-ta2?/Ptr-) and M2354 (Pi-ta+/Pi-ta2-/Ptr), and S/C272 (Pi-ta/Pi-ta2?/Ptr+). Pictures were taken at seven days post-inoculation
Fig. 4
Fig. 4
Ptr gene variation within Oryza species. The DNA sequences from the fourth exon of Ptr in cultivated rice and wild rice relatives were used to construct a phylogenetic tree. All the DNA sequences of the wild rice relatives were downloaded from the Gramene website (www.gramene.org). a Natural variation in Ptr. The haplotype was determined based on five nonsynonymous and 12 bp InDel differences between Katy and Amane in addition to one InDel in front of a 12 bp InDel resulting in a frame-shift of Ptr. Exons are indicated by black filled rectangles and Armadillo (ARM) repeats (Superfamily 1.75) are indicated by the red filled rectangles. In the table, the triangles indicate the insertion and width of triangles indicate the insertion size. The black lines in the triangles indicate that the nonsynonymous substitution occurred inside of the 12 bp. NA means data is not available. b Sliding window of nucleotide variations in the Ptr ORF. The blue rectangles indicate the number and position of each exon. c Phylogenetic tree of Ptr in Oryza species (Supplementary Fig. 8). Hap A corresponds to Pi-ta/Pi-ta2/Ptr-containing varieties and Hap B corresponds to Pi-ta-containing rice varieties. Microscale units are as indicated
Fig. 5
Fig. 5
DNA sequence polymorphism of Ptr in 3 K rice germplasm. a Sliding window analysis of polymorphism with SNPs from indicated coding regions showed in the bars. x-axis is the total number for SNPs in exons. b Phylogenetic tree of each haplotype (Hap) in supplementary Table 7. Hap 16 has an identical Ptr sequence as Katy. Hap 1, Hap 6, Hap 12, Hap 15, and Hap 16 grouped in the pink rectangle contain the identical resistant Pi-ta allele in Katy (see Supplementary Data 2 for information on rice varieties for each haplotype and details of SNPs). Evolutionary distance is as indicated on x-axis

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