A sorghum MYB transcription factor induces 3-deoxyanthocyanidins and enhances resistance against leaf blights in maize

Abstract

Sorghum responds to the ingress of the fungal pathogen Colletotrichum sublineolum through the biosynthesis of 3-deoxyanthocyanidin phytoalexins at the site of primary infection. Biosynthesis of 3-deoxyanthocyanidins in sorghum requires a MYB transcription factor encoded by yellow seed1 (y1), an orthologue of the maize gene pericarp color1 (p1). Maize lines with a functional p1 and flavonoid structural genes do not produce foliar 3-deoxyanthocyanidins in response to fungal ingress. To perform a comparative metabolic analysis of sorghum and maize 3-deoxyanthocyanidin biosynthetic pathways, we developed transgenic maize lines expressing the sorghum y1 gene. In maize, the y1 transgene phenocopied p1-regulated pigment accumulation in the pericarp and cob glumes. LC-MS profiling of fungus-challenged Y1-maize leaves showed induction of 3-deoxyanthocyanidins, specifically luteolinidin. Y1-maize plants also induced constitutive and higher levels of flavonoids in leaves. In response to Colletotrichum graminicola, Y1-maize showed a resistance response.

Figure 1

Schematic representation of the biosynthetic pathway of flavonoid compounds. Enzyme names are (gene names in parentheses): PAL, Phenylalanine ammonia lyase; C4H, Cinnamate-4-hydroxylase; 4CL, 4-coumarate: coenzymeA ligase; C3'H, p-coumarate 3'-hydroxlase; CHS (c2), Chalcone synthase; CHI (chi1), Chalcone isomerase, DFR (a1), Dihydroflavonol reductase; and F3'H, Flavonoid 3'-hydroxylase (pr1). Pathway modeled after [1,21,22,23,24].

Figure 2

Characterization of y1 transgenes. (A) Structural features of the sorghum y1 gene. The gray box represents the upstream regulatory region. The bent arrow indicates the transcription start site. Solid boxes correspond to exons that are joined by angled lines representing introns. The restriction enzyme sites shown are: H, HindIII; K, KpnI; SL, SalI; SC, ScaI. Illustration not drawn to scale. (B) Sorghum y1 gene-induced pigmentation phenotypes in transgenic Y1-maize. Three y1 transgenic events representing Y1-rr, Y1-pr and Y1-wr were characterized for ear, husk, tassel glumes, leaf mid-rib, and silk browning phenotypes. Comparable controls included are: plants segregating for the absence of y1 transgene shown as negative segregant (NS) and native p1 expressing alleles P1-rr and P1-wr and HII (from A188 X B73), used for transformation. (C) Sorghum y1 gene induces flavonoid structural genes in Y1-maize. The expression of the y1 transgene and four flavonoid structural genes relative to the housekeeping gene glyceraldehyde phosphate dehydrogenase was assayed using RT-PCR. Expression was tested in the pericarp tissues of the Y1-rr and Y1-pr transgenes and their respective negative segregants (Y1-rr and Y1-pr). c2: chalcone synthase, chi: chalcone isomerase, a1: dihydroflavonol reductase, pr1: flavonoid 3'-hydroxylase, gapdh: glyceraldehyde phosphate dehydrogenase.

Figure 3

Sorghum y1 gene induces accumulation of flavonoid compounds in transgenic maize leaves. Y1-rr, Y1-pr, and Y1-wr are independent transgenic events; NS, Negative segregant; P1-rr and P1-wr, maize lines carrying endogenous p1 alleles. Values shown are mean ± SE. (A) Total flavonoids expressed as catechin equivalents; (B) Flavan-4-ols expressed as absorbance at 550 nm.

Figure 4

Sorghum y1 gene enhances resistance against C. heterostrophus and C. graminicola in Y1-maize. (A) Detached leaf assay showing disease symptoms that developed 4 days post infection (dpi) when infected with C. heterostrophus. (i) Y1-rr; (ii) Y1-pr; (iii) Y1-wr; (iv) NS; (v) P1-wr; (vi) un-inoculated Y1-pr. Scale bar indicates 1 mm. (B) Quantification of the lesion area 4 dpi with C. heterostrophus. Values shown are the mean ± SE. (C) Symptoms that developed 11 dpi when whole plants were infected with C. graminicola. (D) Quantification of lesion area 11 dpi with C. graminicola. Values shown are the mean of 44 replicates ± SE. The x-axis in Figure 4A,C shows different genotypes used: Y1-rr, Y1-pr, Y1-wr; NS and P1-wr.

Figure 5

Induction of 3-deoxyanthocyanidins and their derivatives in Y1-maize. LC-MS chromatograms obtained from the luteolinidin standard (A), infected leaves of Y1-rr ((B,C) representing two biological replicates), and an overlay (D) of Y1-rr (sample B; blue trace) and NS (pink trace) are presented for comparison. The m/z values of the extracted chromatograms were similar to those of the luteolinidin standard (271.060).