An autoactive NB-LRR gene causes Rht13 dwarfism in wheat

Abstract

Semidwarfing genes have greatly increased wheat yields globally, yet the widely used gibberellin (GA)-insensitive genes Rht-B1b and Rht-D1b have disadvantages for seedling emergence. Use of the GA-sensitive semidwarfing gene Rht13 avoids this pleiotropic effect. Here, we show that Rht13 encodes a nucleotide-binding site/leucine-rich repeat (NB-LRR) gene. A point mutation in the semidwarf Rht-B13b allele autoactivates the NB-LRR gene and causes a height reduction comparable with Rht-B1b and Rht-D1b in diverse genetic backgrounds. The autoactive Rht-B13b allele leads to transcriptional up-regulation of pathogenesis-related genes including class III peroxidases associated with cell wall remodeling. Rht13 represents a new class of reduced height (Rht) gene, unlike other Rht genes, which encode components of the GA signaling or metabolic pathways. This discovery opens avenues to use autoactive NB-LRR genes as semidwarfing genes in a range of crop species, and to apply Rht13 in wheat breeding programs using a perfect genetic marker.

Keywords: Triticum aestivum L. (wheat); autoactive NB-LRR; reduced-height (Rht) gene; semidwarfing gene.

Fig. 1.

Phenotypic characteristics of Magnif (Rht-B13a) and Magnif M (Rht-B13b). (A) Magnif and (B) Magnif M grown under greenhouse conditions at Zadoks growth stage 69. Developmental time-course of (C) stem length and (D) peduncle length in wheat grown under field conditions. Data points combine measurements from 5–10 individual field-grown plants. The error bars represent the SEM.

Fig. 2.

Mapping of the NB-LRR gene Rht13. (A) Rht13 is located on the long arm of chromosome 7B. (B) Physical mapping interval in CDC Stanley with genetic markers (SSR and BAC derived). The distal region (gray box) contained more SNPs between all samples and the reference sequence. (C) SNPs (triangles) and INDEL (circle) between tall and short progeny from a Magnif x Magnif M cross, identified by chrom-seq. The red triangle indicates amino acid change-inducing SNP. (D) Transcripts identified by RNA-seq of progeny from a Magnif x Magnif M cross. The asterisk indicates a significantly differentially expressed transcript between tall and short progeny. (E) Intron–exon structure of gene encoded by Rht13. Exons are represented by boxes, with untranslated regions in pale blue and coding regions in darker blue, and introns are represented by thin gray lines. (F) The gene encodes a 1,272-amino acid protein containing an Rx, NB-ARC, and LRR domain and is annotated as MSTRG.55039 (SI Appendix). Magnif M has a mutation (S240F) in the RNBS-A motif (yellow).

Fig. 3.

Validation that the S240F mutation in Rht-B13b causes a reduction in height. (A) Cadenza0453 segregates for plants homozygous for the wild-type allele Rht-B13a (Left) and mutant allele Rht-B13b (Right) and (B) Cadenza0453 height quantification, the black bars represent the mean, *** P < 0.001, Student’s t test. (C) Height of T₁ progeny of two transgenic events (families 2 and 6) in Fielder background transformed with Rht-B13b allele, stunted plants are represented by points immediately above the X-axis (details in SI Appendix, Table S6). (D) and (E) show families 2 and 6, respectively. Null segregants (-) are on the left of each image.

Fig. 4.

Effect of Rht-B13b and conventional dwarfing alleles Rht-B1b and Rht-D1b on stem and peduncle length in different wheat backgrounds in the field. (A–C) stem length, (D–F) peduncle length, (A and D) EGA Gregory, (B and E) Espada and (C and F) Magenta. Letters indicate significant differences at maturity determined by a one-way ANOVA followed by the Tukey post hoc test (P < 0.05). Data points combine measurements from 5–20 individual field-grown plants. The error bars represent the SEM.

Fig. 5.

Rht-B13b induces defense gene responses in N. benthamiana and wheat. (A) Alignment of the RNBS-A motif from Rht-B13a and Rht-B13b protein with the tomato I-2 protein and the I-2 mutant (S233F) that induces autoactivation. (B) Infiltration of Rht-B13b into N. benthamiana induces significantly more cell death (right side of leaf) than Rht-B13a (left side of leaf, no cell death observed). Black arrows indicate the infiltrated region. The experiment was repeated twice, on six plants each time, a representative result is shown 6 d post inoculation. Expression of PR genes PR3 (C–E) and PR4 (F–H) were measured in wheat basal peduncle (C, F), apical peduncle (D, G), and flag leaf blade (E, H). PR gene expression was normalized to actin. For each graph, the expression level is normalized to be 1 in Rht-B13a, error bars represent the SE (n = 3–4). Significant differences were calculated using a t test on log transformed values, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

Changes in class III peroxidase gene expression, hydrogen peroxide content, mechanical and cell properties in mutant (Rht-B13b) compared with wild-type (Rht-B13a) peduncles. (A–E) are in a Magnif background, (F) and (G) are in a Cadenza background. (A) Fold change in expression of 218 class III peroxidase genes compared with an equivalent number of randomly selected genes. Purple dots represent genes differentially expressed at padj < 0.001 with a fold change >2, gray dots are not differentially expressed, lines across the violin plot represent quartile 1, the median and quartile 3. (B) Hydrogen peroxide content in elongating peduncles. Significant differences determined by Student’s t test, n = 6. Peduncle bending strength (C) and bending rigidity (D) were determined using a three-point bend test, significant differences were determined using Student’s t tests, n = 11–12. (E) Epidermal cell lengths in inter-hair and single cells, significant differences determined by ANOVA, n = 62–190 individual cells. (F) and (G) transverse sections imaged with bright-field illumination (magnification 20X) from the apical peduncle (AP) 1 cm below the ear, the peduncle midpoint (MP) and the basal peduncle (BP) 1 cm above the node. (F) is stained with toluidine blue O and (G) with phloroglucinol-HCl. One representative image from five independent biological replicates is shown. Asterisks indicate statistical differences between genotypes: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.

Model of pathway through which Rht-B13b causes semidwarfism. In a wild-type plant (Rht-B13a, Left) the NB-LRR protein is inactive resulting in normal cell wall cross-linking, cell expansion, and growth. In the autoactive mutant (Rht-B13b, Right), PR genes including class III peroxidases are up-regulated in expanding tissues. Class III peroxidases may use H₂O₂ to increase cell wall cross-linking, which results in reduced cell expansion and growth.