The monosaccharide transporter gene, AtSTP4, and the cell-wall invertase, Atbetafruct1, are induced in Arabidopsis during infection with the fungal biotroph Erysiphe cichoracearum

Abstract

Powdery mildew fungi are biotrophic pathogens that form a complex interface, the haustorium, between the host plant and the parasite. The pathogen acts as an additional sink, competing with host sinks, resulting in considerable modification of photoassimilate production and partitioning within the host tissue. Here, we examine the factors that may contribute to these changes. We show for the first time in one biotrophic interaction (Arabidopsis/Erysiphe cichoracearum) all of the following responses: Glc uptake in host tissues is enhanced after fungal infection; this coincides with the induction of expression of the monosaccharide transporter gene, Arabidopsis sugar transport protein 4 (AtSTP4), in infected leaves; invertase activity and transcript levels for a cell wall invertase, Atbetafruct1, increase substantially in Arabidopsis during attack by this pathogen. Before infection, Arabidopsis plants transformed with an AtSTP4 promoter-beta-glucuronidase construct show expression mainly in sink tissues such as roots; after infection, AtSTP4 expression is induced in the mature leaves and increases over the 6-d time period. Sections of infected leaves stained for beta-glucuronidase show that AtSTP4 expression is not confined to infected epidermal cells but is also evident in a wider range of cells, including those of the vascular tissue. The results are discussed in relation to the possible coordinated expression of hexose transporters and cell wall invertase in the host response to powdery mildew infection.

Figure 1.

Glc uptake into Arabidopsis source leaves after infection with E. cichoracearum. Glc uptake into infected (brushed with inoculum) and noninfected (brushed without inoculum) Arabidopsis leaf discs was measured using 0.5 mm [¹⁴C]Glc (1, noninfected; 2, infected). Treatments 2 and 4 were 6 d post inoculation. Measurements were also taken after removal of the mycelium with sellotape before uptake (3, noninfected; 4, infected). Results are the mean (±se) of three experiments, and the asterisk indicates significant difference from Glc uptake in corresponding control noninfected leaf discs (P ≤ 0.001).

Figure 2.

Northern analysis of AtSTP4 expression after infection with E. cichoracearum. a, Forty micrograms of total RNA per lane was separated on a 1.2% (w/v) agarose gel and transferred to a nylon membrane. Hybridization was carried out with a digoxigenin (DIG)labeled AtSTP4 (top) and actin (bottom) probes. Blots were washed at high stringency. Tissue was treated with fungal inoculum by gently brushing with a paintbrush. Controls were brushed without inoculum. Lane 1, Noninfected, non-brushed tissue (d 0); lane 2, noninfected brushed tissue (d 0); lane 3, control tissue (d 3); lane 4, infected tissue (d 3); lane 5, control tissue (d 6); and lane 6, infected tissue (d 6). A representative blot is shown. The experiment was performed three times for AtSTP4 and twice for actin. Densitometry data were obtained for each experiment and the mean band intensity is shown for AtSTP4 (b) and actin (c).

Figure 3.

Histochemical analysis of mature transgenic Arabidopsis leaves expressing the GUS gene under the control of the AtSTP4 promoter after inoculation with compatible and incompatible mildew strains. a, To visualize the location of GUS activity (indicated by blue color), leaves were incubated at 22°C for 15 h in the presence of 5-bromo-4-chloro-3-indolyl-β-d-glucuronide-sodium salt trihydrate. Results are shown for 1, 3, and 6 d postinoculation for (i), healthy, uninfected leaf; (ii), leaf with the incompatible B. graminis; and (iii), leaf with the compatible E. cichoracearum. b, Tissue was stained for GUS then fixed, embedded, and sectioned. Cell walls are stained pink with Schiff reagent. (i), Control healthy leaf; (ii), +6 d postinoculation with E. cichoracearum. UE, Upper epidermis; LE, lower epidermis; VB, vascular bundle. Representative leaves for each condition are shown.

Figure 4.

Northern analysis of AtSTP3 expression after infection with E. cichoracearum. a, Twenty-five micrograms of total RNA per lane was separated on a 1.5% (w/v) agarose gel and transferred to a nylon membrane. Hybridization was carried out with a ³²P-labeled AtSTP3 probe (top). Methylene blue staining of the 25s rRNA band is also shown (bottom). Blots were washed at moderate stringency. Tissue was treated with fungal inoculum by gently brushing with a paintbrush. Controls were brushed without inoculum. Lane 1, Noninfected tissue (d 0); lane 2, infected tissue (d 0); lane 3, control tissue (d 1); lane 4, infected tissue (d 1); lane 5, control tissue (d 3); lane 6, infected tissue (d 3); lane 7, control tissue (d 6); and lane 8, infected tissue (d 6). A representative blot is shown. The experiment was performed three times with similar results. Densitometry data were obtained for two experiments, and the mean band intensity is shown for AtSTP3 (b) and 25S rRNA (c).

Figure 5.

Effect of infection on cell wall invertase activity in Arabidopsis source leaves. Invertase activity was assayed at pH 4.5 in extracts isolated from leaves treated with (black bars) or without (white bars) fungal inoculum. Noninfected control tissue was gently brushed without fungal inoculum. Values are the means of four replicated experiments. Asterisk indicates significant difference from control plants (P ≤ 0.001).

Figure 6.

Effect of infection on invertase expression in Arabidopsis source leaf using RT-PCR. a, RT-PCR was carried out with primers designed to generate partial-length invertase (top) or actin cDNAs (bottom). Products were run on a 1.2% (w/v) agarose gel, hybridized with DIG-labeled Atβfruct1 and actin cDNA probes, and washed at high stringency. Lane 1, Noninfected (d 0); lane 2, infected (d 0); lane 3 noninfected (d 1); lane 4, infected (d 1); lane 5, noninfected (d 3); lane 6, infected (d 3); lane 7, noninfected (d 6); and lane 8, infected (d 6). b and c, Densitometry data were obtained for each amplified product and expressed relative to the products at d 0 (healthy), which were normalized to a value of 1. The data are from a representative experiment repeated twice.