Alterations in sterol homeostasis in erp1 plants
The identification of PSAT1 as the gene affected in the erp1 mutant suggests a role of sterol esters for pathogen-inducible responses of Arabidopsis. The enzyme encoded by ERP1, PSAT1, catalyzes the formation of sterol esters by transferring acyl groups from the sn2 position of phosphatidylethanolamine to sterols, as determined with microsomal fractions from PSAT1-overexpressing Arabidopsis plants (Banas et al., 2005). Although a function of the second product of the PSAT reaction, lysophosphatidylethanolamine, cannot be ruled out, it is generally assumed that the loss of sterol ester formation is responsible for phenotypes in mutants with impaired PSAT activity (Banas et al., 2005; Bouvier-Navé et al., 2010). Postulated functions for sterol esters so far include their role as a transport or storage form of sterols (Dyas and Goad, 1993). As such, they might be involved in sterol sequestration if more sterols are present in the cell than required. This has been shown in a tobacco mutant with a 10-fold higher sterol content, where excess sterols accumulated as sterol esters and were compartmentalized in cytoplasmic lipid bodies (Maillot-Vernier et al., 1991; Gondet et al., 1994), as well as in tobacco plants overproducing sterols as a result of the expression of a cDNA encoding the sterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase (Schaller et al., 1995). In accordance with these observations, the lack of sterol esters in erp1 mutant plants correlates with the absence of Nile Red-stainable lipid bodies (Figure 3b).
The determination of the levels of free sterols, sterol glycosides and acylated sterol glycosides in erp1 mutants revealed a role of PSAT1 in sterol homeostasis. The inability of erp1 plants to synthesize sterol esters correlates with reduced levels of acylated sterol glycosides and enhanced levels of sterol glycosides (Figure 3a). In Arabidopsis, sterol glycosides are synthesized by the UDP-glucose:sterol glucosyltransferases (SGTs) UGT80A2 and UGT80B1, and are degraded by sterol glycoside hydrolases (Grille et al., 2010). In a double knock-out mutant of the two SGT genes (UGT80A2 and B1) , highly reduced levels of sterol glycosides and acylated sterol glycosides correlate with increased free sterol and sterol ester levels in inflorescences and siliques, suggesting a regulatory relationship between the levels of free sterols and conjugated sterols (DeBolt et al., 2009). Genes encoding enzymes catalyzing the synthesis of acylated sterol glycosides, on the other hand, are not known (Grille et al., 2010). Both PSAT1 and the elusive sterol glycoside acyltransferase require phosphoglycerolipids as acyl donors (Banas et al., 2005; Grille et al., 2010). The concomitant decrease of sterol esters and acylated sterol glycosides might suggest a direct involvement of PSAT1 in acylated sterol glycoside synthesis; however, PSAT1 acylates the 3β-OH group of sterols, whereas acylated sterol glycosides are acylated at the C6 of glucose.
Several Arabidopsis sterol biosynthesis mutants exhibit strong defects in embryonic patterning or growth, which demonstrates the importance of a well balanced phytosterol content (reviewed in Schaller, 2003). Despite the strong reduction of sterol ester levels in erp1 mutant plants and concomitant alterations in the quantity of other sterol lipids, the molecular species composition of free sterols, sterol glycosides and acylated sterol glycosides is comparable with those determined in leaves of wild-type plants (Figures S5 and S7). Thus, a lack of PSAT1 activity does not affect sterol biosynthesis in general. In accordance with this, erp1 mutant plants show no developmental defects when grown under optimal conditions, except for the early leaf senescence. These findings, together with the observation of reduced growth in growth competition experiments of the are1 are2 yeast mutant (Zweytick et al., 2000), which is impaired in sterol ester synthesis, indicate the functional importance of sterol ester formation during developmental aging and stress adaptation.
Role of sterol esters in the pathogen response
Our genetic screen aimed at the identification of genes involved in non-host resistance to P. infestans. In the pathogen-challenged erp1 mutant, the lack of sterol ester accumulation correlates with an enhanced number of mesophyll cells undergoing cell death, and with the formation of massive callose depositions in the mesophyll cell layer. This aberrant response was observed after infection by the non-adapted pathogens P. infestans and Bgh, as well as by the host-adapted pathogen G. cichoracearum. The enhanced response does not correlate with obvious alterations in the growth of the pathogen. Thus, we did not observe significantly enhanced growth of P. infestans in the erp1 mutants compared with their respective wild types (Figure 1), nor did we detect enhanced oomycete colonization of the mesophyll cell layer. Similarly, there were no differences in cell death and the number of entry sites in response to Bgh inoculation (Figure 5). These observations show that the deregulated callose depositions are not a consequence of enhanced growth and entry attempts of pathogens in the mesophyll cell layer. It should rather be postulated that a diffusible signal induces the aberrant callose depositions in the mesophyll cell layer. This diffusible signal would originate from the primary infection attempts of the filamentous pathogens in the epidermal cell layer. The nature of this signal is not known. It might be speculated that reactive oxygen species generated at the infection sites contribute to this cell non-autonomous signal; however, these also accumulate in response to bacterial infections (Torres et al., 2006), and do not induce the formation of aberrant callose depositions (Figure 6). As the massive callose depositions in the mesophyll cell layer are formed in response to inoculation with invasive filamentous pathogens, but not after bacterial infection, it is possible that damage-induced molecular patterns, generated by the invading pathogen, might act as signaling molecules.
The transient silencing of a sterol biosynthesis gene in tobacco resulted in enhanced membrane permeability, as deduced from increased staining of nuclei with a membrane-impermeable dye (Wang et al., 2012). Assuming that the altered sterol homeostasis in erp1 plants also leads to alterations in the sterol content of plasma membranes, it is thus possible that erp1 epidermal cells release larger quantities of the proposed diffusible signal(s).
Alternatively, erp1 mesophyll cells might have an enhanced responsiveness to the putative diffusible signal(s). Phytosterols constitute structural components of plasma membranes, which modulate acyl chain ordering and water permeability (Schuler et al., 1991). In particular, membrane microdomains, which are defined as heterogenous and highly dynamic areas of the plasma membrane, are enriched in sterols and sphingolipids (Mongrand et al., 2004). Free sterols and sterol glycosides have been reported to occur in detergent-resistant membranes (DRMs) isolated from Arabidopsis seedlings (Laloi et al., 2007). Thus, lateral membrane compartments are supposed to provide platforms for signaling events (Mongrand et al., 2010). A role of sterols for plant pathogen responses might be concluded from the observation that Filipin staining of barley leaf epidermal cells infected with Bgh shows an enrichment of sterols at sites of pathogen attack (Bhat et al., 2005). In Arabidopis, enhanced levels of stigmasterol in pathogen-infected leaves (Griebel and Zeier, 2010; Wang et al., 2012; Figure S6) lead to alterations in membrane composition (Griebel and Zeier, 2010). As stigmasterol stimulates H+-ATPase activity in plasma membranes in vitro (Grandmougin-Ferjani et al., 1997), increased stigmasterol levels might have a direct effect on membrane function. We thus hypothesize that the altered sterol homeostasis in erp1 mutant plants might have an impact on cellular signaling events in response to pathogen infection.
The massive accumulation of callose in response to infection with P. infestans might be the result of de novo synthesis or the inhibition of callose degradation, a mechanism that has been suggested to regulate callose accumulation at plasmodesmata (Zavaliev et al., 2011). The callose synthase-like protein GSL5, responsible for wound- and pathogen-induced callose synthesis (Jacobs et al., 2003; Nishimura et al., 2003), is not required for erp1-associated callose deposition (Figure 2). Interestingly, the non-adapted barley pathogen Bgh can penetrate pmr4 mutants slightly better than the wild type, suggesting that GSL5 has a function in entry resistance in the non-host resistance of Arabidopsis against Bgh (Jacobs et al., 2003). GSL5 has also been shown to be required for callose deposition in response to inoculation with the non-adapted pathogen Colletotrichum truncatum (Shimada et al., 2006). A second callose synthase gene, GSL6, is activated in response to treatment with salicylic acid (SA) , as well as after infection with Hyaloperonospora parasitica in an NPR1-dependent manner (Dong et al., 2008). Whether GSL6 plays a role in callose synthesis in erp1 plants remains to be determined. In GSL6-RNAi plants, callose formation in response to pathogen infection is not impaired (Jacobs et al., 2003). Microarray studies do not show major changes in GSL5 or GSL6 gene expression in response to P. infestans (http://www.genevestigator.com). Non-host resistance against Bgh does not involve changes in GSL6, and only minor changes in GSL5 expression (http://www.genevestigator.com).
In conclusion, the identification of PSAT1 as a gene involved in the response of Arabidopsis to infection by the non-adapted pathogens P. infestans and Bgh, as well as the adapted powdery mildew pathogen, G. cichoracearum, suggests a previously unreported role of sterol conjugates for pathogen defense responses in Arabidopsis.