Amino acid changes during sunflower infection by the necrotrophic fungus B. cinerea

Abstract

Metabolic changes that occur in host tissues during a necrotrophic plant/fungal interaction have been poorly investigated. Whereas carbon metabolism reprogramming and photosynthesis disturbances have been studied,¹ data on plant amino acids stores during infection are scarce. Here we report an analysis of sunflower cotyledon amino acid content during infection with the necrotrophic fungus Botrytis cinerea, by using ¹³C-NMR spectroscopy. A rapid disappearance of plant amino acids was observed, most probably due to fungal assimilation. In order to explore amino acid changes due to host reaction, we investigated the amino acid content in healthy and invaded region of infected leaves. During the course of infection, glutamate store was affected at distance in the non invaded region. Glutamate depletion was correlated to an enhanced sunflower glutamate dehydrogenase (GDH) transcription level in the area invaded by pathogen. Our data suggest that glutamate could be transferred to the invaded region to supply nitrogen. Such a strategy could delay cell death, and consequently disturb fungal progression in plant tissues.

As a necrotrophic fungus, Botrytis cinerea induces host cell death to enable rapid colonization of plants and derive nutrients from sacrificed cells.^2,3 Main pathogenicity factors, toxin and lytic enzyme secretion act synergistically to kill, degrade and macerate host plant tissues.⁴ Oxalic acid secretion by Sclerotinia sclerotiorum is now considered as a subtle process to promote plant defence mechanisms that will contribute to host cell death and favor pathogen to progress and feed.⁵

We have investigated soluble carbon transfer from host to pathogen using ¹³C-NMR spectroscopy during sunflower cotyledon infection by B. cinerea.⁶ Plant soluble sugars (glucose, fructose and sucrose) rapidly disappeared and were converted to fungal metabolites (mannitol, trehalose and glycogen), generating a strong carbohydrate capacity. Mannitol pathway plays a central role in B. cinerea carbon metabolism. This pathway allows glucose and fructose conversion into mannitol by independent routes, fructose being essentially converted in mannitol.⁶

Soluble carbon metabolite profiling, performed through the course of infection, revealed also the fate of amino acids from plant and fungal origin. Here we present amino acid changes during infection of sunflower cotyledon. PCA extracts obtained from healthy eight-days-old sunflower germlings, infected cotyledons (24, 48, 72, 94 and 120 hpi) and B. cinerea mycelium collected after saprophytic growth, were analyzed by ¹³C-NMR spectroscopy (Fig. 1). Glutamine, glutamate, arginine and asparagine (0.7, 0.4, 0.4 and 0.2 mg.g⁻¹ FW) were the main amino acid detected in healthy sunflower cotyledons. In B. cinerea mycelium, the main amino acids detected by NMR spectroscopy were glutamate, glutamine, aspartic acid, arginine and alanine (0.9, 0.4, 0.2, 0.16, 0.08 mg.g⁻¹ FW). In fungal mycelium and cotyledon, glutamate and glutamine were the main amino acid stores. During infection, amino acid concentration drastically decreased to be negligible 72 hpi. Between 0 and 48 hpi, glutamine, glutamate and arginine concentration decreased by 70, 50 and 70% respectively, whereas asparagine concentration decreased by 40% only (Fig. 1). Our results suggest that plant amino acids could be consumed by B. cinerea during infection. Moreover, asparagine could be less favorably metabolized than other amino acids. However, experiments conducted on sucrose starvation of sycamore cells revealed asparagine accumulation.⁷ During infection of sunflower by B. cinerea sucrose disappear rapidly.⁶ Asparagine persistence in infected sunflower tissues could then be explained by its accumulation in host cells rather than fungal consumption. Decrease in amino acid during a necrotrophic infection is in agreement with earlier observations on B. cinerea/tomato leaves and S. sclerotiorum/sunflower cotyledons interactions.^8,9 For sure, a pathogenic fungus is expected to drain nutrient from a plant. However, the concentration of most plant derived amino acids and total nitrogen content of the leaf apoplast increased during the Cladosporium fulvum/tomato hemibiotrophic compatible interaction.¹⁰ One possibility mentioned for amino acid increase during infection could be an increase in apoplastic protease activity, probably a serine protease, induced in tomato upon infection. The fungus could therefore manipulate plant metabolism to maintain or increase the apoplastic concentration of nitrogen compound.¹¹ This must be particularly the case for biotrophs and hemibiotrophs fungal pathogens that derive nutrients from living plant cells.

Figure 1.

¹³C-NMR spectra of perchloric acid (Pca) extracts of B. cinerea mycelium, Helianthus annuus cotyledons and sunflower cotyledons infected by B. cinerea 48 and 120 hpi. NMR experiments, fungal strain, plant growth and plant infection were performed as described previously.⁵ Three independent experiments were done from separated infection series and cultures. Representative spectra are presented here.

Moreover, we cannot exclude that amino acid concentration could be modified by host metabolism changes, induced by the necrotrophic pathogen. To adress this question, we evaluated the impact of infection on healthy areas of infected leaves. For this purpose, we analyzed separately, by NMR spectroscopy, infected and non infected regions of cotyledons at 48 hpi (half of the cotyledons were colonized by B. cinerea, Fig. 2B). Glutamate storage was affected at distance during sunflower infection by B. cinerea. Its concentration was decreased by 35% in the non-invaded region of leaves, beyond the infected area. Control cotyledons, maintained in the same growth conditions as inoculated cotyledons for a period of time equivalent to the course of infection, did not reveal significant changes. Similarly, a 40% decrease in glutamate was observed 24 hpi, in the non invaded region of cotyledon infected by S. sclerotiorum.⁸ Glutamate dehydrogenase (GDH), a senescence-related marker, could be a component of the plant defence response during plant-pathogen interaction, related to cell death.¹² GDH transcription was activated during infection of sunflower by the necrotrophic fungus Phoma macdonadii and by fungal elicitors.^12,13 It has been suggested that GDH expression could be regarded as a cell survival process during the late phase preceding cell death.¹² We analyzed, by real time Q-PCR the expression of a GDH transcript sunflower EST (TC16502, http://compbio.dfci.harvard.edu/index.html). Whereas GDH transcription level was high in the invaded area of infected cotyledons, its expression was very low in the non invaded region (Fig. 2). This result suggests that glutamate depletion, detected in healthy region could be the effect of a plant defense reaction activated in invaded areas. Amino acid and ammonium content of the infected area were then investigated. A 50 to 80% amino acid decrease (according to amino acid), and a 50% ammonium decrease were detected 48 hpi, as compared to control leaves. Glutamate could be transferred from healthy to invaded region, to supply nitrogen to this dying area. Glutamate supply could also contribute to slow down cell death in order to hamper progression of the pathogen.

Figure 2.

Localized expression of sunflower glutamate dehydrogenase gene (GDH) during infection by B. cinerea. (a) expression of a GDH sunflower EST during infection by B. cinerea. Total RNA was isolated from healthy and infected plant tissues collected 24, 48, 72, 96 hpi. For 24 and 48 hpi, cotyledons were separated in healthy (H) or infected (i) areas. expression analysis was performed by quantitative RT-PCR, as described previously,⁵ using GDH primers (forward primer, CCTTGG TGT CTTATG TCG GATTT; reverse primer, ACCTCT GGATGG TAT CTG ATTCCT) and was calibrated to EF1α gene¹¹ (forward primer, AAG GCC GAG CGT GAAAGA G; reverse primer, GTG CAG TAG TACTTG GTG GTCTCAA). Three independent cDNA preparations obtained from independent infected and healthy cotyledon biological replicates of were used for analysis. Bars represent standard deviation. Data above histogram bars indicate glutamate concentration (mg.g⁻¹ FW). (B) representative infection sequence (from 0 to 96 hpi), H and I represent healthy and infected area of infected cotyledons, respectively.

The present study highlights the impact of a fungal necrotrophic infection on plant amino acids metabolism. Our results suggest that B. cinerea could act as a nitrogen sink during infection. Moreover, by redistributing nitrogen stores, host plant could delay cell death, essential for necrotrophic growth.

Abbreviations

hpi

hour post infection

PCA

perchloric acid

FW

fresh weight

Addendum to: Dulermo T, Rascle C, Chinnici G, Gout E, Bligny R, Cotton P. Dynamic carbon transfer during pathogenesis of sunflower by the necrotrophic Fungus Botrytis cinerea: from plant hexoses to mannitol. New Phytol. 2009 doi: 10.1111/j.1469-8137.2009.02890.x. In press.