Flavonoid phytoalexin-dependent resistance to anthracnose leaf blight requires a functional yellow seed1 in Sorghum bicolor

Abstract

In Sorghum bicolor, a group of phytoalexins are induced at the site of infection by Colletotrichum sublineolum, the anthracnose fungus. These compounds, classified as 3-deoxyanthocyanidins, have structural similarities to the precursors of phlobaphenes. Sorghum yellow seed1 (y1) encodes a MYB transcription factor that regulates phlobaphene biosynthesis. Using the candystripe1 transposon mutagenesis system in sorghum, we have isolated functional revertants as well as loss-of-function alleles of y1. These near-isogenic lines of sorghum show that, compared to functionally revertant alleles, loss of y1 lines do not accumulate phlobaphenes. Molecular characterization of two null y1 alleles shows a partial internal deletion in the y1 sequence. These null alleles, designated as y1-ww1 and y1-ww4, do not accumulate 3-deoxyanthocyanidins when challenged with the nonpathogenic fungus Cochliobolus heterostrophus. Further, as compared to the wild-type allele, both y1-ww1 and y1-ww4 show greater susceptibility to the pathogenic fungus C. sublineolum. In fungal-inoculated wild-type seedlings, y1 and its target flavonoid structural genes are coordinately expressed. However, in y1-ww1 and y1-ww4 seedlings where y1 is not expressed, steady-state transcripts of its target genes could not be detected. Cosegregation analysis showed that the functional y1 gene is genetically linked with resistance to C. sublineolum. Overall results demonstrate that the accumulation of sorghum 3-deoxyanthocyanidin phytoalexins and resistance to C. sublineolum in sorghum require a functional y1 gene.

Figure 1.—

Flavonoid biosynthetic pathway of anthocyanins, phlobaphenes, and 3-deoxyanthocyanidins in sorghum. Phenylalanine undergoes a series of enzymatic steps to give rise to 4-coumaroyl-CoA. CHS then catalyzes the stepwise condensation of one molecule of coumaroyl-CoA and three molecules of malonyl-CoA as the first step in the flavonoid biosynthetic pathway to form naringenin chalcone. This then undergoes stereo-specific isomerization by CHI to form naringenin, the common precursor of anthocyanins, phlobaphenes, and 3-deoxyanthocyanidins. The fate of naringenin is determined by the genetic constitution of the plant and the environmental conditions. CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol reductase; F3′ H, flavonoid 3′ hydroxylase. (B) Seed pericarp and mature leaf phenotypes of sorghum genetic stocks carrying Y1-cs30, Y1-rr3, and y1-ww1 alleles.

Figure 2.—

Molecular structure of the y1-ww1 and y1-ww4 alleles. (A) DNA gel blot analysis of Y1-rr3 (R), y1-ww1 (W), and their common progenitor Y1-cs30 (C lanes). A genomic DNA gel blot was hybridized with either an intron II-specific fragment of Y1-rr (F-3) or a y1-cDNA probe. Molecular weight markers in kilobase pairs are shown between the blots. (B) PCR reactions were carried out using genomic DNA as templates and two sets of primer pairs: I, YF-1 and YR-1; II, YF-2 and YR-2. The positions of these primers are shown in C. Lanes are the following: 1, Y1-cs30; 2, Y1-rr3; 3, y1-ww1; and 4, y1-ww4. (C) Line diagram of the structure of y1 in Y1-rr3, y1-ww1, and y1-ww4. A bent arrow represents the transcription start site. Solid boxes are exons joined by angled lines representing introns. The cs1 transposon inserted in intron II is shown as an inverted triangle. The open triangle in the maps of y1-ww1 and y1-ww4 represents the remnants of cs1. The dashed line in y1-ww1 and y1-ww4 indicates sequence deletion. The restriction enzyme sites shown are the following: B, BamHI; E, EcoRI; H, HindIII; K, KpnI; SA, SacI; SL, SalI; SC, ScaI; X, XhoI. The accession numbers corresponding to y1 and cs1 sequences are AY860968 and AF206660, respectively.

Figure 2.—

Molecular structure of the y1-ww1 and y1-ww4 alleles. (A) DNA gel blot analysis of Y1-rr3 (R), y1-ww1 (W), and their common progenitor Y1-cs30 (C lanes). A genomic DNA gel blot was hybridized with either an intron II-specific fragment of Y1-rr (F-3) or a y1-cDNA probe. Molecular weight markers in kilobase pairs are shown between the blots. (B) PCR reactions were carried out using genomic DNA as templates and two sets of primer pairs: I, YF-1 and YR-1; II, YF-2 and YR-2. The positions of these primers are shown in C. Lanes are the following: 1, Y1-cs30; 2, Y1-rr3; 3, y1-ww1; and 4, y1-ww4. (C) Line diagram of the structure of y1 in Y1-rr3, y1-ww1, and y1-ww4. A bent arrow represents the transcription start site. Solid boxes are exons joined by angled lines representing introns. The cs1 transposon inserted in intron II is shown as an inverted triangle. The open triangle in the maps of y1-ww1 and y1-ww4 represents the remnants of cs1. The dashed line in y1-ww1 and y1-ww4 indicates sequence deletion. The restriction enzyme sites shown are the following: B, BamHI; E, EcoRI; H, HindIII; K, KpnI; SA, SacI; SL, SalI; SC, ScaI; X, XhoI. The accession numbers corresponding to y1 and cs1 sequences are AY860968 and AF206660, respectively.

Figure 3.—

Comparative response of etiolated seedlings of Y1-rr3 and y1-ww1 after inoculation with C. heterostrophus. (A) Accumulation of reddish-brown phytoalexin compounds in Y1-rr3 and y1-ww1 mesocotyls at 0, 24, and 36 hpi with C. heterostrophus. The upper mesocotyl in each panel is from the Y1-rr3 allele while the lower one is from the y1-ww1 allele. (B) TLC analysis of methanolic extracts prepared from inoculated Y1-rr3 and y1-ww1 mesocotyls. Luteolinidin and apigeninidin were loaded as standards.

Figure 4.—

Spectrophotometry of induced compounds. Quantification of flavonoid compounds from methanolic extracts prepared from Y1-rr3 and y1-ww1 mesocotyls harvested at 0, 24, and 36 hpi. Absorbance was recorded at 480 nm. The error bars represent the standard error of three replicates.

Figure 5.—

Characterization of the 3-deoxyanthocyanidins from inoculated mesocotyls of Y1-rr3 and y1-ww1 in response to C. heterostrophus. (A) HPLC chromatograms monitored at 480 nm. Pure luteolinidin and apigeninidin were eluted at ∼10 and 11 min, respectively. (B) LC-MS profile of the Y1-rr3 extract shows three major and a number of minor ions. The major ions at 269.1, 271.1, and 285.2 m/z correspond to methoxy-apigeninidin (MA), luteolinidin (L), and methoxy-luteolinidin (ML), respectively. One of the minor ions at 255 m/z corresponds to apigeninidin (A), while other minor ions have not been identified. The profile of the y1-ww1 extract shows two minor peaks with mass/charge ratios of 269.1 and 271.1.

Figure 6.—

Gene expression analysis of y1 and the flavonoid structural genes. (A) Slot blot hybridization of radioactively labeled first-strand cDNA from RNA isolated 24 hpi. “Empty” means no DNA was added as a control for background hybridization. (B) Temporal expression of y1 and flavonoid structural genes in Y1-rr3 mesocotyls after inoculation with C. heterostrophus. Blots were probed with cDNA of y1, chalcone synthase (chs1), chalcone isomerase (chi1), flavonoid 3′ hydroxylase (f3′h3), and dihydroflavonol reductase (dfr1). Numbers at the top represent hours after inoculation with C. heterostrophus.

Figure 7.—

Fungal infection and plant responses after infection with C. sublineolum. (A) Inoculated leaves of the three genotypes showing disease symptoms 4 dpi (a, c, and e) and 11 dpi (b, d, and f). Restriction of the disease is shown in the Y1-rr3 (b). Development of characteristic ALB symptoms with necrosis of the leaf as the lesions grow and coalesce is shown in the null y1-ww lines (d and f). (B) Quantification of lesion density at 4 dpi (I), percentage of infection area at 11 dpi (II), and sporulation at 8 dpi (III). (C) HPLC analysis of the induced 3-deoxyanthocyanidin compounds 72 hpi with C. sublineolum. (Top) A chromatogram of pigments extracted from lesions of inoculated Y1-rr3 leaves showing the accumulation of luteolinidin (L, retention time 13.63 min), apigeninidin (A, 14.2 min), and 7-methoxy-apigeninidin (MA, 16.2 min). (Bottom) A chromatogram of pigments extracted from inoculated plants of the y1-ww1 allele.

Figure 8.—

Cosegregation analysis of disease phenotype and y1 gene in a testcross population of [(Y1-rr3 × BT×623) × BT×623]. (A) ALB phenotypes of C. sublineolum-infected leaves of Y1-rr3 and BT×623 observed at 7 dpi. (B) Genotype and disease phenotype of testcross progeny plants. “R/B” lanes indicate Y1-rr3/BT×623 heterozygotes, and “B” lanes are homozygous for BT×623. The disease phenotype corresponding to each plant is shown below the lane.