Defence Mechanism Of Arabidopsis And Tomato To Sphinganine-analog Mycotoxins

Fumonisin and AAL-Toxin are sphinganine-analog mycotoxins (SAMT), which induce reactive oxygen species (ROS) and then promote programmed cell death (PCD) by disrupting the sphingolipids metabolism. Fumonisin could contaminate asparagus and various Brassica vegetables while AAL-Toxin causes Alternaria stem canker on susceptible tomato cultivars. Thus, to clarify the mechanism of plant defence aganist SAMT will deepen our understanding of plant defence system, and help us to control the contamination of SAMT on various vegetables through chemical regulation and genetics engineering. To elucidate the specific defence mechanism in Cruciferae, the present study investigated the roles of secondary metabolite glucosinolates and defensive phytohormone jasmonic acid (JA) in plant defence against SAMT-induced PCD and their mechanism in Arabidopsis. Furthermore, the gene against fumonisin B1(FBI) was cloned from FBI-resistant mutant fbr41, then was used to transform to susceptible tomato cultivars to analyse the sensitivity of transgenic tomato to AAL-Toxin, which will deepen our understanding on plant defence mechanism to SAMT-induced PCD, as well as provide a effective way to enhance tomato resistance to SAMT-induced PCD, and to breed for tomato variety resistant to Alternaria stem cancer. The results are as follow:1、In order to clarify the specific defence mechanism in Cruciferae, the effect of glucosinolates in FBI-induced PCD were studied in Arabidopsis. The results showed that tryptophan-derived secondary metabolic pathways were stimulated upon FBI infiltration. The expression of genes involved in indolic glucosinolate and camalexin biosynthesis pathways were induced. By contrast, FBI infiltration suppressed the expression of genes involved in aliphatic glucosinolates biosynthesis. The accumulation of indolic and aliphatic glucosinolates was both reduced in FBI infiltrated leaves, suggesting that the biosynthesis and hydrolysis of indolic glucosinolates were both activated in FB1-induced PCD response. The phenotypic assay of glucosinolate-related mutants showed that indolic glucosinolate-deficient mutant cyp79B2cyp79B3and classical myrosinase-deficient mutant tgg1tgg2exhibited severer lesions than wild-type while aliphatic glucosinolate-deficient mutant myb28myb29, camalexin-null mutant pad3-1and atypical myrosinase-deficient mutant pen2-2exhibited wild-type lesion response upon FBI infiltration. All above data indicated that TGG-dependent hydrolysis of indolic glucosinolates is involved in Arabidopsis defence to FB1-induced PCD. Exogenous application of intact indolic glucosinolates and their breakdown products, indolic-3-carbinol (I3C) and indolic-3-acetonitrile (IAN) attenuated FB1-induced PCD on Col-0and cyp79B2cyp79B3leaves. In addition, exogenous application of I3C and I3A reduced FBI-induced ROS accumulation in Col-0and cyp79B2cyp79B3leaves, which made us conclude that TGG-dependent indolic glucosinolates hydrolysis attenuated FBI-induced PCD through reducing the FB1-induced ROS accumulation.2、The effect of jasmonic acid (JA) signaling on PCD induced by FB1in intact Arabidopsis seedlings and its mechanism were investigated using JA-deficient and JA-insensitive mutants. The results indicated that both JA-deficient and JA-insensitive mutants exhibited more serious lesions than wild-type while the exogenous application of JA reduced lesion formation in Col-0, suggesting that JA plays a negative role in FBI-induced cell death. The glucosinolate profiles showed that indolic glucosinolates accumulation in all JA-related mutants were significantly reduced relative to Col-0, while the application of JA dramatically induced not only indolic glucosinolates level but also I3C and IAN as well as myrosinase activity, indicating that there might exist a correlation between the level of indolic glucosinolates and sensitivity to FBI with JA application. A phenotypic assay was performed in Col-0and indolic glucosinolate-deficient mutant cyp79B2cyp79B3seedlings with JA-pretreatment. The results showed that although JA pretreatment reduced the formation of lesions in cyp79B2cyp79B3, the mutant still exhibited much more severe lesion phenotype than Col-0, which indicated that the prevention of FB1-induced cell death by JA pretreatment is partly dependent on indolic glucosinolates biosynthesis. 3、The mutant gene causing the FBI-resistant phenotype in fbr41was cloned and analysed by forward genetics. Genetic analysis revealed that fbr41phenotype was controlled by a single dominate nuclear locus, and mapped-based cloning results showed that there is a mutation form G to A in intron of LCB2b/FBR41in fbr41. In order to distinguish LCB2b/FBR41in wild-type and mutant, LCB2b/FBR41in fbr41was termed as FBR41-D. We found that the mutation site located in the first base of intron which was important for spliceosome to recognize the introns, and then excise them from the pre-mRNA to produce the translatable mRNA. Thus the mutation may cause disorder in cutting. Sequencing data verified that there was a insertion of ATTTT in FBR41-D cDNA sequence which caused the change of reading frame after the insertion site. According to the analysis of amino acid sequence translated from the FBR41-D cDNA, we found that the termination codon was formed earlier than it did in translation of LCB2b/FBR41, which stopped the translation of FBR41-D in advance. A molecular complementation experiment showed that Col-0achieved FB1-resistance after FBR41-D transformation, indicating that FBR41-D caused the FBI-resistance phenotype. To analyse the function of FBR41-D will clarify our understanding on plant defence mechanism and benefit us to enhance vegetable resistance to SAMT through genetics engineering.4、AAL-Toxin causes Alternaria stem canker on susceptible tomato cultivars, which is need to be controlled in tomato production. FBR41-D from Arabidopsis was transformed to susceptible tomato cultivars Castlemart (CM), and to analyse its role in tomato sensitivity to AAL-Toxin and Alternaria stem canker resistance. The results showed that35S::FBR41-D transgenic tomato exhibited less lesions than CM, which formed a number of lesions. Moreover, H2O2staining indicated that H2O2was accumulated in AAL-Toxin treated CM leaves while the H2O2accumulation was suppressed in transgenic tomato, suggesting that susceptible tomato cultivars CM became resistant to AAL-Toxin after transformation, which maybe rely on the inhibition of FBR41-D to serine palmitoyltransferase (SPT), the first enzyme in sphingolipids biosynthesis pathway. All above data indicated that FBR41-D exerted broad-spectrum resistant to SAMT and the activity of tomato SPT directly affects tomato sensitivity to AAL-Toxin, which provide a theoretical base for further research on tomato defense mechanism to SAMT as well as molecular resisatance breeding for Alternaria stem canker.