Maize susceptibility to Ustilago maydis is influenced by genetic and chemical perturbation of carbohydrate allocation

Abstract

The ability of biotrophic fungi to metabolically adapt to the host environment is a critical factor in fungal diseases of crop plants. In this study, we analysed the transcriptome of maize tumours induced by Ustilago maydis to identify key features underlying metabolic shifts during disease. Among other metabolic changes, this analysis highlighted modifications during infection in the transcriptional regulation of carbohydrate allocation and starch metabolism. We confirmed the relevance of these changes by establishing that symptom development was altered in an id1 (indeterminate1) mutant that showed increased accumulation of sucrose as well as being defective in the vegetative to reproductive transition. We further established the relevance of specific metabolic functions related to carbohydrate allocation by assaying disease in su1 (sugary1) mutant plants with altered starch metabolism and in plants treated with glucose, sucrose and silver nitrate during infection. We propose that specific regulatory and metabolic changes influence the balance between susceptibility and resistance by altering carbon allocation to promote fungal growth or to influence plant defence. Taken together, these studies reveal key aspects of metabolism that are critical for biotrophic adaptation during the maize-U. maydis interaction.

Keywords: RNA-Seq; Zea mays; maize; smut; starch; transcription factor.

Figure 1

Zea mays gene expression in seedlings, tassels and ears in tumours induced by Ustilago maydis. The id1, why1 and su1 genes (in the box outlined with a solid line) showed constant gene expression in tumours, but with developmental expression changes. The bd1 gene (in the box outlined with the dotted line) showed unchanged expression independent of the developmental stage of the plant or infection status. The gn1, ra2, lg3 and bad1 genes (in the box outlined with the broken line) showed differential gene expression dependent on developmental state and infection state. The data were derived from three independent replicates. Standard error is shown. *P ≤ 0.05; **P ≤ 0.01; ns, not significant.

Figure 2

Enriched biological gene ontology (GO) term networks of Zea mays infected with Ustilago maydis (10 days post‐inoculation) for down‐regulated genes. More general GO terms are indicated by larger circles and the intensity of the red colour indicates the significance level of GO term enrichment based on the P value (see Table S4) for the GO term.

Figure 3

Enriched biological gene ontology (GO) term networks of Zea mays infected with Ustilago maydis (10 days post‐inoculation) for up‐regulated genes. More general GO terms are indicated by larger circles and the intensity of the red colour indicates the significance level of GO term enrichment based on the P value (see Table S4) for the GO term.

Figure 4

Categorization of the most up‐ and down‐regulated host genes in Ustilago maydis‐induced tumours at 10 days post‐inoculation (dpi). (A, C, E) Up‐regulated genes. (B, D, F) Down‐regulated genes. (A, B) Known transcripts from http://www.maizegdb.org; (C, D) transcripts from Golden Bantam; (E, F) genes from the classical gene list. n represents the number of genes in each chart. Numbers after the different categories indicate the number of genes in this category. The colour coding between the different charts signifies the same classification.

Figure 5

The transcription factor ID1 is important for the infection success of Ustilago maydis. The id1 homozygous (id1/id1) and heterozygous (Id1/id1) mutant lines and the corresponding wild‐type (wt) (Id1/Id1) line were infected with U. maydis. The infection progression was scored at 14 days post‐inoculation (dpi) and the data of three biological replicates are shown. Red pigment formation was observed in severely infected wt plants, which showed stem tumours or were dead (inset shows a close up view of the plant from the left photograph), compared with green homozygous id1/id1 mutant plants of the same infection class (inset shows a close up view of the plant from the right photograph). Standard deviations are shown and asterisk indicates a significant difference at P < 0.05 for the disease index (DI). DI was calculated for each biological replicate based on the following scoring scheme for symptoms: 0, healthy plants/no symptoms; 1, anthocyanin formation; 2, leaf tumours; 3, small stem tumours; 4, big stem tumours; 5, plant death. The overall percentages of plants of the three repeats in each category are indicated in the bar graphs and correlate with DI.

Figure 6

Sucrose and glucose supplementation increases the virulence of Ustilago maydis. Seedlings (14 days old) were infected with U. maydis and daily injections with water, sucrose or glucose were applied after the infection was established (see Experimental Procedures for details). (A) The plants were scored for disease symptoms (DI, disease index) after 14 days of infection and treatment as described in Fig. 5. (B) The glucose‐treated control plants (70.2%) showed signs of chlorosis near the injection sites, as shown in the photograph on the right. **Significant difference at P < 0.01. Standard deviations are shown.

Figure 7

Silver treatment induces anthocyanin production and reduces disease progression in maize. (A) Untreated control plants showed unimpaired growth with little anthocyanin formation in the stem area. (B) Plants treated with 0.412 g AgNO₃ per litre of soil showed reduced growth and increased anthocyanin production in the stems and basal parts of the leaves. Note that the plants were photographed at the same magnification in (A) and (B). (C) Disease symptoms and disease rating (DI, disease index) of untreated, but infected, control plants after 14 days. (D) Disease symptoms and disease rating of infected and AgNO₃ (0.412 g/L soil)‐treated plants after 14 days. Plants were infected as 7‐day‐old seedlings with a mating‐compatible culture of wt strains at 5 × 10⁶ cells/mL. AgNO₃ was added to the soil at the beginning of the experiment. The experiment was repeated three times and with a range of additional silver concentrations (Fig. S3). Standard deviations are shown. **Statistically significant differences at P < 0.01. The plant symptoms were scored as described in Fig. 5.

Figure 8

Carbohydrate metabolism of the host is changed during infection and is implicated in disease progression. (A) Infected and uninfected stem tissues at 10 days post‐inoculation (dpi) were stained for starch, and starch kernels were visually identified in infected and uninfected stem tissue. (B) Starch content of uninfected and infected stem tissue was determined as reducing sugars. (C) Disease symptom progression and disease index (DI) ratings for the su1/su1 mutant line and the maize wild‐type (wt; Su1/Su1) line at 14 dpi infected with Ustilago maydis. Standard deviations are shown. **Significant difference at P < 0.01. The plant symptoms were scored as described in Fig. 5.