Chloroplast-associated metabolic functions influence the susceptibility of maize to Ustilago maydis

Abstract

Biotrophic fungal pathogens must evade or suppress plant defence responses to establish a compatible interaction in living host tissue. In addition, metabolic changes during disease reflect both the impact of nutrient acquisition by the fungus to support proliferation and the integration of metabolism with the plant defence response. In this study, we used transcriptome analyses to predict that the chloroplast and associated functions are important for symptom formation by the biotrophic fungus Ustilago maydis on maize. We tested our prediction by examining the impact on disease of a genetic defect (whirly1) in chloroplast function. In addition, we examined whether disease was influenced by inhibition of glutamine synthetase by glufosinate (impacting amino acid biosynthesis) or inhibition of 3-phosphoshikimate 1-carboxyvinyltransferase by glyphosate (influencing secondary metabolism). All of these perturbations increased the severity of disease, thus suggesting a contribution to resistance. Overall, these findings provide a framework for understanding the components of host metabolism that benefit the plant versus the pathogen during a biotrophic interaction. They also reinforce the emerging importance of the chloroplast as a mediator of plant defence.

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

Figure 1

Chloroplast functions are important for the infection success of Ustilago maydis. (A) A U. maydis induced tumour in the stem area of a Zea mays plant at 16 days post‐infection (dpi). The pigments were extracted with ethanol (80%) from uninfected and infected stem sections at 10 dpi. (B) Thin layer chromatography (TLC) was performed with pigments extracted from 500 mg of uninfected and infected samples at 10 dpi. RF, retention factor. The heading ‘[%] of uninfected’ is the intensity of the infected spot divided by the intensity of the uninfected spot expressed as a percentage. (C) The results of seedling infection studies with the wild‐type (wt) and why1‐2/why1‐2 mutant infected with U. maydis wt cells. The infection test was performed in triplicate. DI, disease index. *P < 0.05. Standard deviations are shown.

Figure 2

Inhibition of secondary metabolism increases maize susceptibility to Ustilago maydis. Glyphosate (Roundup^®) is an inhibitor of 3‐phosphoshikimate 1‐carboxyvinyltransferase (EPSPS), leading to reduced chorismate production. Treatment with more than a 1 : 20 dilution of the recommended dose of 10 mL/L led to plant death. Treatment with 1 : 30 or 1 : 40 of the recommended dose allowed plant survival with symptoms such as reduced chlorophyll and stunted growth and tiller formation (A). A 48‐h pretreatment before infection with U. maydis led to increased disease symptoms and higher disease index (DI). Multiple tumours on one plant were found (B). The infection studies were repeated three times with different concentrations. *P < 0.05. Standard deviations are shown.

Figure 3

Disrupted amino acid metabolism increases maize susceptibility to Ustilago maydis. Glufosinate (Ignite^®) is an inhibitor of plant glutamine synthetases. Treated plants were killed at the full recommended dose (5 mL/L), but survived the treatment with one‐fifth, one‐tenth and one‐twentieth of the recommended dose, with minor visible plant deformations (A). Seedlings that were pretreated with glufosinate (48 h) and subsequently infected with U. maydis for 14 days showed increased disease symptoms and had a higher disease index (DI) with increasing glufosinate dose (B). Infection of the plants for 5 days and consecutive treatment with one‐tenth of the recommended dose led to increased disease symptoms and a higher DI (C). The infection tests were performed three times. Significance levels: *P < 0.05; **P < 0.01. Standard deviations are shown.