Resistance of callose synthase activity to free fatty acid inhibition as an indicator of Fusarium head blight resistance in wheat

Abstract

The fungal pathogen Fusarium graminearum is the causal agent of Fusarium head blight (FHB); a devastating crop disease resulting in heavy yield losses and grain contamination with mycotoxins. We recently showed that the secreted lipase FGL1, a virulence factor of F. graminearum, targets plant defense-related callose biosynthesis during wheat head infection. This effector-like function is based on a FGL1-mediated release of polyunsaturated free fatty acids (FFA) that can inhibit callose synthase activity. The importance of FGL1 in successful wheat head colonization was demonstrated in FGL1 disruption mutants (Δfgl1), where infection was restricted to directly inoculated spikelets and accompanied by strong callose deposition in the spikelet’s phloem. The application of polyunsaturated FFA to Δfgl1-infected spikelets prevented callose deposition in the phloem and partially restored wheat head colonization.

 

The comparative analysis of 3 wheat cultivars revealed that the level of resistance to FHB correlated with resistance to FFA-dependent inhibition of callose biosynthesis. Therefore, resistance of callose biosynthesis to FFA inhibition might be used as marker and/or direct target in the breeding of FHB-resistant wheat cultivars.

Fusarium head blight (FHB) of wheat is one of the most destructive crop diseases worldwide.^1-3 The causal agent of FHB, the necrotrophic fungal plant-pathogen Fusarium graminearum enters the wheat (Triticum aestivum) head via flowering spikelets from where it colonizes the whole spike, resulting in drastic yield losses. The remaining grain can also be contaminated by the mycotoxin deoxynivalenol (DON) and not be used for food or feed production. F. graminearum produces DON during infection where it is required as a virulence factor for complete colonization of the wheat head and induction of FHB disease symptoms.^4,5 Therefore, epidemic outbreaks of FHB can have a direct impact on food security as wheat is one of the predominantly cultivated crops in temperate climates according to the 2012 FAO statistics of crop production (FAOSTAT of the Food and Agriculture Organization of the United Nations). Because of the importance of F. graminearum in agriculture and science, this fungus was classified as a Top 10 plant pathogen.⁶

Only a few wheat cultivars were identified showing FHB resistance, but these cultivars revealed inappropriate agronomic traits making them unsuitable for commercial breeding approaches.⁷ One of the wheat cultivars with an increased FHB resistance is Sumai 3. In contrast to susceptible wheat cultivars, Sumai 3 showed an increased deposition of the (1,3)-β-glucan cell wall polymer callose in the transition zone of the spikelet’s rachilla and rachis.⁸ This callose deposition correlated with an increased spreading resistance, which has been referred to as type II resistance,⁹ after initial F. graminearum infection. In general, callose deposition at sites of pathogen penetration contributes to the plant’s innate immunity¹⁰ and has been considered as a physical barrier to slow pathogen invasion.^11,12 In our recent studies with the model plant Arabidopsis thaliana, we could show that callose can also completely stop fungal penetration if deposited in elevated amount at early time-points of infection.¹³

Similar to the callose deposition in Sumai 3, we observed callose deposition in the phloem of the spikelet’s transition zone in the FHB-susceptible wheat cultivar Nandu (Lochow-Petkus, Bergen-Wohlde, Germany) after infection with the F. graminearum disruption mutant Δfgl1, but not after infection with the respective wild-type strain.¹⁴ In the Δfgl1 strain, the secreted lipase FGL1 was disrupted, which resulted in a strongly reduced virulence of the fungus. Unlike wild-type, Δfgl1 was unable to fully colonize the wheat head, and infection was restricted to the directly inoculated spikelet.^14,15 Comparing the infection process in wild-type- and Δfgl1-infected wheat spikes with microscopic and biochemical methods, we were able to unravel the role of FGL1 in F. graminearum virulence. Analysis of the free fatty acid (FFA) content of infected spikelet tissue revealed that the secreted lipase FGL1 is required to release relatively high amounts of the polyunsaturated FFAs linoleic and linolenic acid. Based on in vitro and in planta assays that showed a high capacity of callose biosynthesis inhibition by these FFAs, we concluded that FGL1-mediated inhibition of plant defense-related callose deposition in the spikelet’s transition zone is a decisive factor in breaking this layer of the wheat’s type II resistance.¹⁴ These results also suggest that a higher resistance of the wheat’s callose synthases to FFA inhibition may support type II resistance to F. graminearum.

To test whether differences in susceptibility of callose biosynthesis to FFA inhibition would exist in different wheat cultivars and might correlate with the strength of FHB symptoms, we compared the cultivars Nandu, Thasos (Strube, Söllingen, Germany), and Batis (Strube) in pathogenicity tests with F. graminearum wild-type.¹⁵ At 21 d post-inoculation of the wheat head, we observed strongest FHB symptoms on Nandu, where the complete head was necrotic and bleached, followed by Thasos, where about 2/3 of the head revealed strong disease symptoms. In contrast, the wheat cultivar Batis showed relatively weak disease symptoms only in the area of initial fungal point-inoculation (Fig. 1A). Statistical analysis of disease symptoms confirmed these observations with about 90% infected spikelets for Nandu, which was similar to our recent results for this wheat cultivar,¹⁴ 68% for Thasos, and 23% for Batis (Fig. 1B). In the following assays, we compared callose synthase activity in membranes, which were isolated from wheat head tissue.¹⁷ After FFA treatment at concentrations ranging from 0.7 to 700 µM,¹⁴ stearic acid (18:0; x:y denotes a fatty acid with x carbons and y double bonds) affected callose synthase activity in none of the cultivars. Palmitic acid (16:0) inhibited callose synthase activity only in the cultivars Nandu and Thasos at high concentrations (Fig. 1C). However, a strong inhibitory effect in all cultivars was detected for the polyunsaturated FFAs linoleic (18:2) and linolenic acid (18:3) that revealed a predominant role in inhibiting defense-related callose deposition in the wheat spike.¹⁴ At a concentration of 17.5 µM, we determined an activity reduction of 25% in Batis, nearly 50% in Thasos, and almost 80% in Nandu. Complete inhibition of callose synthase activity due to application of linoleic and linolenic acid was observed at a concentration of 35 µM for Nandu. At this concentration, a residual activity of almost 30% was detected for Batis and 15% for Thasos. Activity was completely inhibited by polyunsaturated FFAs at a concentration of 70 µM in all cultivars (Fig. 1C). The inhibitory effect of the monounsaturated oleic acid (18:1) was not as strong as of the polyunsaturated FFAs (Fig. 1C). The relative callose synthase activity after addition of linolenic acid reflects the generally higher resistance of the cultivar Batis in comparison to Thasos and Nandu toward application of strongly callose synthase-inhibiting FFA (Fig. 1D).

Figure 1. Susceptibility against F. graminearum infection and callose synthase activity of the wheat cultivars Nandu, Thasos, and Batis after free fatty acid application. (A) Two central spikelets were each inoculated with 10 µl of water (c) and 200 conidia of F. graminearum wild-type (wt). Arrows indicate inoculation site. Spikes are representative for disease phenotype at 21 d post-inoculation (dpi). (B) Table indicating susceptibility of the tested wheat cultivars against F. graminearum infection (F.g. wt). Repeat experiments gave similar results (a). Infection referred to partially or completely bleached spikelets observed 21 dpi. Spikelets showing minor symptoms (tiny yellow or brown spots) were not counted. Results are the average of 15 inoculated wheat heads (16–24 spikelets per head) inoculated with 400 conidia (b). Values statistically different from each other at P < 0.05 Tukey test, n = 15. (C) Callose synthase activity of membranes isolated from untreated spikes of wheat cultivars Nandu, Thasos, and Batis 7 d after anthesis (Zadoks stages 7.5–7.9).¹⁶ FFAs dissolved in ethanol added to the membranes reaching final concentrations of 0.7, 7, 17.5, 35, 70, and 700 µM in the reaction buffer. FFA: palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), α-linolenic acid (18:3). Values represent the mean of 2 biologically independent experiments. a, b, c, d: P < 0.05 Tukey test. Error bars represent ± SEM, and n = 6. (D) Percent callose synthase activity of the cultivars Nandu and Thasos relative to cultivar Batis after addition of linolenic acid (18:3) at indicated concentrations.

Comparing the results of the pathogenicity tests with the callose synthase activity assays of the 3 wheat cultivars, we observed that the severity of FHB disease symptoms correlated with the strength of FFA-induced inhibition of callose biosynthesis. Our recent findings showed that callose-related type II resistance is a target of FGL1-mediated FFA release by F. graminearum.¹⁴ Therefore, a higher resistance of callose biosynthesis to pathogen-induced FFA inhibition would support the establishment of this layer of type II resistance in the wheat head. The extent to which resistance of callose biosynthesis to FFA inhibition might be used in marker assisted breeding, could be tested in future experiments. Molecular breeding approaches would also allow targeting callose biosynthesis directly. The expression of additional callose synthases with a possibly higher resistance to FFA inhibition might be a strategy to increase FHB resistance in cereals attacked by F. graminearum.