Secreted Fungal Effector Lipase Releases Free Fatty Acids to Inhibit Innate Immunity-Related Callose Formation during Wheat Head Infection

The deposition of the (1,3)-β-glucan cell wall polymer callose at sites of attempted penetration is a common plant defense response to intruding pathogens and part of the plant’s innate immunity. Infection of the Fusarium graminearum disruption mutant Δfgl1, which lacks the effector lipase FGL1, is restricted to inoculated wheat (Triticum aestivum) spikelets, whereas the wild-type strain colonized the whole wheat spike. Our studies here were aimed at analyzing the role of FGL1 in establishing full F. graminearum virulence. Confocal laser-scanning microscopy revealed that the Δfgl1 mutant strongly induced the deposition of spot-like callose patches in vascular bundles of directly inoculated spikelets, while these callose deposits were not observed in infections by the wild type. Elevated concentrations of the polyunsaturated free fatty acids (FFAs) linoleic and α-linolenic acid, which we detected in F. graminearum wild type-infected wheat spike tissue compared with Δfgl1-infected tissue, provided clear evidence for a suggested function of FGL1 in suppressing callose biosynthesis. These FFAs not only inhibited plant callose biosynthesis in vitro and in planta but also partially restored virulence to the Δfgl1 mutant when applied during infection of wheat spikelets. Additional FFA analysis confirmed that the purified effector lipase FGL1 was sufficient to release linoleic and α-linolenic acids from wheat spike tissue. We concluded that these two FFAs have a major function in the suppression of the innate immunity-related callose biosynthesis and, hence, the progress of F. graminearum wheat infection.The molecular and physiological regulation of the biosynthesis of callose, which is a (1,3)-β-glucan polymer with some (1,6)-branches (Aspinall and Kessler, 1957), and its importance for plant development as well as plant defense are still under examination. Regarding the involvement of callose in plant defense responses, particular attention has been focused on the formation of cell wall thickenings in plants, so-called papillae, at sites of microbial attack. They were already described 150 years ago (deBary, 1863) and reported to commonly contain callose (Mangin, 1895). Since then, examinations have identified callose as the most abundant chemical constituent in papillae, which may also include proteins (e.g. peroxidases and antimicrobial thionins), phenolics, and other constituents (Aist and Williams, 1971; Sherwood and Vance, 1976; Mims et al., 2000). Papillae have been regarded as an early defense reaction that may not completely stop the pathogen; rather, they have been considered to act as a physical barrier to slow pathogen invasion (Stone and Clarke, 1992; Voigt and Somerville, 2009) and to contribute to the plant’s innate immunity (Jones and Dangl, 2006; Schwessinger and Ronald, 2012). The host plant can gain time to initiate defense reactions that require gene activation and expression, such as the hypersensitive reactions, phytoalexin production, and pathogenesis-related protein synthesis (Lamb and Dixon, 1997; Brown et al., 1998). However, our recent study revealed that callose can also act as a barrier that completely prevents fungal penetration. The overexpression of POWDERY MILDEW RESISTANT4 (PMR4), a gene encoding a stress-induced callose synthase, resulted in early elevated callose deposition at sites of attempted powdery mildew penetration in Arabidopsis (Arabidopsis thaliana; Ellinger et al., 2013). Interestingly, the pmr4 deletion mutant also showed an increased resistance to powdery mildew that, however, was induced at later stages of powdery mildew infection because an initial fungal penetration still occurred. In fact, the absence of the functional callose synthase PMR4 in the pmr4 mutant resulted in papillae that were free from callose but also induced a hyperactivation of the salicylic acid defense pathway, which was shown to be the basis of resistance in double mutant and microarray analyses (Jacobs et al., 2003; Nishimura et al., 2003). The callose synthase gene PMR4 from Arabidopsis belongs to the GLUCAN SYNTHASE-LIKE (GSL) family, genes that have been identified in higher plants including wheat (Triticum aestivum; Cui et al., 2001; Doblin et al., 2001; Hong et al., 2001; Østergaard et al., 2002; Voigt et al., 2006). The predicted function of these genes as callose synthases is generally supported by homology with the yeast FK506 SENSITIVITY (FKS) genes, which are believed to be subunits of (1,3)-β-glucan synthase complexes (Douglas et al., 1994; Dijkgraaf et al., 2002). Additionally, the predicted proteins encoded by the GSL genes correlate with the approximately 200-kD catalytic subunit of putative callose synthases. Li et al. (2003) showed that the amino acid sequence predicted from a GSL gene in barley (Hordeum vulgare; HvGSL1) correlates with the amino acid sequence of an active (1,3)-β-glucan synthase fraction.In this study, we aimed to examine the involvement of callose synthesis and callose deposition in plant defense against intruding fungal pathogens in the pathosystem wheat-Fusarium graminearum. We focused on the ability of wheat to inhibit a further spread of fungal pathogens after an initial, successful infection. This resistance to fungal spread within the host has been referred to as type II resistance and is part of a widely accepted two-component system of resistance, which includes type I resistance operating against initial infection (Schroeder and Christensen, 1963). For our analyses, we used the direct interaction between wheat as host and F. graminearum as a pathogen. On the one hand, Fusarium head blight (FHB) of wheat, caused by F. graminearum, is one of the most destructive crop diseases worldwide (McMullen et al., 1997; del Blanco et al., 2003; Madgwick et al., 2011) and classifies this fungus as a top 10 plant pathogen based on its importance in science and agriculture (Dean et al., 2012). On the other hand, only a limited number of wheat cultivars were identified that revealed FHB resistance. However, these cultivars did not qualify for commercial cultivation or breeding approaches due to inappropriate agronomic traits (Buerstmayr et al., 2009). Further elucidation of the mechanisms of spreading resistance could support the generation of FHB-resistant wheat cultivars.In this regard, we demonstrated that the secreted lipase FGL1 of F. graminearum is a virulence factor required for wheat infection (Voigt et al., 2005). A strong resistance to fungal spread was observed in a susceptible wheat cultivar after infection with the lipase-deficient F. graminearum strain Δfgl1. Light microscopy indicated barrier formation in the transition zone of rachilla and rachis of directly inoculated spikelets. In contrast, neither spreading resistance nor barrier formation was observed during F. graminearum wild type infection. An active role of lipases in establishing full virulence was also recently proposed for the plant pathogen Fusarium oxysporum f. sp. lycopersici, where reduced lipolytic activity due to the deletion of lipase regulatory genes resulted in reduced colonization of tomato (Solanum lycopersicum) plants (Bravo-Ruiz et al., 2013). Because the expression of the lipase-encoding gene LIP1 was induced in the biotrophic fungus Blumeria graminis during early stages of infection (Feng et al., 2009) and disruption of the putative secreted lipase gene lipA resulted in reduced virulence of the bacterial plant pathogen Xanthomonas campestris (Tamir-Ariel et al., 2012), a general importance of extracellular lipolytic activity during plant colonization is indicated.We evaluated a possible role of callose in plant defense by infecting wheat spikes with the virulent fungal pathogen F. graminearum wild type, the virulence-deficient F. graminearum deletion mutant Δfgl1, and the barley leaf pathogen Pyrenophora teres, the latter intended to induce strong plant defense responses as known from incompatible, nonhost interactions. The formation of callose plugs within the vascular bundles of inoculated spikelets and the callose synthase activity of infected spikelet tissue correlated directly with increased plant resistance. Subsequent analyses of free fatty acid (FFA) concentrations revealed that those polyunsaturated FFAs were enriched during wheat infection with the F. graminearum wild-type strain that could inhibit callose synthase activity in vitro as well as in planta and partially restored the virulence of the lipase-deficient F. graminearum strain Δfgl1. On the basis of these results, we propose a model for FHB where defense-related callose synthase is inhibited by specific FFAs whose accumulation is caused by the fungus during fungal infection; this inhibition is required for full infection of the wheat head.