The Lr34 adult plant rust resistance gene provides seedling resistance in durum wheat without senescence

Abstract

The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad-spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field-grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome-encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up-regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress-response genes were up-regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad-spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.

Keywords: Blumeria; Puccinia; Triticum; ABC transporter; rust.

Figure 1

Lr34 durum resistance corresponds to transgene expression levels. (a) Chitin assay quantification of P. triticina growth on wheat seedlings (three‐ to four‐leaf stage) of hexaploid cultivar Thatcher (Th), a near‐isogenic Lr34 Thatcher line (Th+34), Stewart (St), Lr34 lines 17‐1, 36‐4, 39‐2, 41‐2 and uninfected Thatcher (Th‐ve). Seedlings were grown at 10 °C (black columns) or 22 °C (white columns) and harvested at 30 and 14 dpi, respectively. Common letters (white columns) or numbers (black columns) indicate data not significantly different (ANOVA, P < 0.05), throughout. Each value is the average of four chitin measurements from 10 to 15 pooled seedlings. (b) Lr34 expression was quantified by Q‐PCR and normalized relative to GAPDH in uninfected, three‐ to four‐leaf seedlings of genotypes described in (a). Each data point is derived from three biological replicates, each with three technical replicates. (c) Chitin assay quantification of P. striiformis f. sp. tritici growth (14 dpi) on 10‐15 seedlings per genotype described in (a) at the three‐ to four‐leaf stage. Using pairwise t‐tests (P < 0.05), line 39‐2 had significantly less pathogen growth than lines 17‐1 and 41‐2, which each had less growth than line 36‐4. A nontransgenic Stewart line regenerated from tissue culture (null) is included.

Figure 2

Phenotypic analysis of Lr34 durum seedlings. (a) P. triticina growth (22 °C, 16‐h light) 14 dpi on leaves of seedling of Stewart, Lr34 lines 17‐1, 39‐2, 41‐2 and 36‐4, Thatcher hexaploid wheat and Lr34 Thatcher. (b) P. striiformis f. sp. tritici growth (22 °C, 16‐h light) 14 dpi on Lr34 seedlings of 17‐1, 39‐2 and 41‐2, uninfected Stewart and infected Stewart. Microscopy of P. triticina growth (12 dpi) on Stewart (c) and Lr34 line 39‐2 (d) stained with WGA‐FITC. (e) P. triticina growth (12dpi) on Lr34 line 39‐2 showing one moderate and numerous small infection sites. (f) P. triticina haustoria (arrows) in cells of line 39‐2. (g, h) P. striiformis f. sp. tritici growth on Stewart (g) and Lr34 line 39‐2 (h) (WGA‐FITC stained). 3′, 3′‐Diaminobenzidine staining of P. triticina infection sites on Stewart (i) and line 39‐2. (j). A brown precipitate shows H₂O₂ accumulation in vascular tissue and cells surrounding uredinia (white circle of cells with yellow centre) in each line. Leaves, in descending order of age (i.e. flag leaf at bottom), from glasshouse‐grown Stewart (k), 39‐2 (l) and 41‐2 (m) plants. (n) Flag leaves from field‐grown (top to bottom) Stewart and lines 17‐1, 39‐2 and 41‐2. Tissues in panels c–j were grown at 22 °C, 16‐h light.

Figure 3

Further characterization of Lr34 durum lines. (a) Chitin assay quantification of Blumeria graminis growth (7 dpi) on leaves of Stewart, 17‐1 and 39‐2. Each data point is from 14 plants and three technical replicates. Fluorescence was converted to ug of chitin/gm of tissue using a standard curve. Data were compared using the Student's t‐test, P < 0.05. (b) Relative Lr34 expression in uninfected (black columns) and P. striiformis f. sp. tritici‐infected (white columns) hexaploid wheat seedlings grown at 10 or 22 °C. Each data point was from pooled leaf tissue (6‐12 seedlings) and triplicate Q‐PCRs. (c) P. striiformis chitin assay quantification (26 dpi) of Stewart, 17‐1, 36‐4, 39‐2, 41‐2 and a nontransgenic Stewart line regenerated from tissue culture (null). Seedlings infected at the three‐ to four‐leaf stage were grown at 22 °C (16‐h light) and tissue, when harvested, was undergoing age‐related senescence (Figure S5). Uninfected Stewart seedlings (un St) were included. Each data point was from pooled leaf tissue of 12 seedlings and four technical replicates. (d) Ltn on flag leaves of field‐grown Lr34 durum lines and Stewart. The length of Ltn was divided by total flag leaf length to calculate % Ltn; 10–26 flag leaves were measured per line (see image 2n).

Figure 4

Lr34 induced gene expression changes in uninfected wheat seedlings. Q‐PCR analyses on RNA from uninfected wheat seedlings (four‐to five‐leaf stage) of hexaploid wheat cultivar Thatcher (Th), a near‐isogenic Lr34 Thatcher line (Th+Lr34), Stewart and Lr34 lines 17‐1, 36‐4, 39‐2 and 41‐2. Panels a–d show relative expression of (a) S40, (b) Cp‐MIII , (c) Rab15 and (d) HSP90, each normalized relative to GAPDH . Data were derived from three biological replicates per genotype and three technical replicates per sample. In panel (d), transgenic lines 17‐1, 39‐2 and 41‐2 had significantly higher levels of gene expression (t‐test, P < 0.05) than the Stewart control, indicated by an asterisk above each column.

Figure 5

Pathogenesis‐related ( PR ) gene expression in Lr34 durum seedlings. (a) Chitin assay quantification of uninfected (black columns) and P. triticina‐infected (12 dpi) (white columns) Stewart seedlings (St) and lines 17‐1, 39‐2 and 41‐2 (10–15 seedlings per genotype). The same plants were used for RNA extraction in (b). (b) PR‐1 gene expression in wheat seedlings normalized relative to the GAPDH . RNA was extracted from uninfected (black columns) or P. triticina‐infected (12 dpi) seedlings (three‐ to four‐leaf stage) described in (a). Three biological replicates were used per genotype and three technical replicates per sample. The same RNAs were quantified for PR‐2 and PR‐3 expression (Figure S7). (c) Relative PR gene expression values (in B and Figure S7) were divided by chitin biomass values of rust‐infected material shown in A. PR1, PR2 and PR3 values are shown as grey, white and black columns, respectively, for Stewart (St) and Lr34 transgenic lines 17‐1, 39‐2 and 41‐2.

Figure 6

Lr34 resistance is influenced by photoperiod, but not associated with H₂O₂ accumulation. (a) Quantification of P. triticina growth (14 dpi) on Stewart (St), Lr34 lines 17‐1, 39‐2, 41‐2 and a nontransgenic tissue culture regenerant (null) under two photoperiod regimes. Seedlings (four‐leaf stage), grown at 22 °C with a 16‐h light/8‐h dark photoperiod), were infected with P. triticina and either returned to these growth conditions (black bars) or grown under constant light (white bars), using the same temperature regime. Each data point is from pooled tissue of approximately six seedlings. (b) Relative H₂O₂ levels, determined by Amplex Red assay, in hexaploid wheat cultivar Thatcher (Th), near‐isogenic Lr34 Thatcher (Th+34), Stewart (St) and Lr34 durum lines 17‐1, 39‐2 and 41‐2 from infected (white column) or uninfected (black column) plants. Each data point is derived from individual H₂O₂ measurements of three biological replicates per genotype.

Figure 7

Lr34 expression and phenotype models. (a) Lr34 resistance requires a minimum transcriptional threshold (expression levels are depicted by arrow width), which is not reached in hexaploid wheat until later in plant maturity. In contrast, Lr34 durum seedlings, Lr34 barley seedlings and cold‐treated, infected hexaploid Lr34 seedlings are resistant due to higher Lr34 expression levels. Lr34 senescence, or leaf tip necrosis (Ltn) (shown in red), is mechanistically the same pathway as resistance but also dependent upon developmental signals (blue arrow labelled DS) that occur later in plant maturation. Hence, cold‐grown, infected Lr34 hexaploid seedlings and Lr34 durum seedlings do not show leaf senescence, while adult plants of the same genotypes do. In Lr34 barley seedlings, signalling during normal developmental senescence of seedling leaves (yellow arrow SS) is sufficient to induce accelerated necrosis due to the heterologous nature of the wheat Lr34 gene. (b) This model is based upon the same assumptions except that Lr34 senescence is considered an independent pathway to resistance.