抗毒素Camalexin对大白菜抗根肿病的作用研究

对易感根肿病大白菜自交系‘BJN3-1’在接种根肿菌(Plasmodiophora brassicae)的同时施用了5个不同剂量的抗毒素camalexin进行处理,研究camalexin在大白菜中抗根肿菌的作用。结果表明,接种根肿菌的对照材料感病症状明显,施用不同剂量camalexin的5个处理在前21 d可明显抑制根肿菌的增殖,随着camalexin施用量的增加对根肿菌生长的抑制作用增强。茉莉酸、水杨酸、乙烯合成途径和病程相关蛋白基因在camalexin处理14~21 d后表达显著高于对照处理。这表明外源施用camalexin能够诱导寄主响应根肿菌胁迫,从而提高抗病性。施用camalexin后28d各处理均严重感病,camalexin对根肿菌增殖的抑制作用减弱,这可能与根肿菌的解毒机制有关。

Arabidopsis Acetyl-Amido Synthetase GH3.5 Involvement in Camalexin Biosynthesis through Conjugation of Indole-3-Carboxylic Acid and Cysteine and Upregulation of Camalexin Biosynthesis Genes

Camalexin （3-thiazol-2＇-yl-indole） is the major phytoalexin found in Arabidopsis thaliana. Several key intermediates and corresponding enzymes have been identified in camalexin biosynthesis through mutant screening and biochemical experiments. Camalexin is formed when indole-3-acetonitrile （IAN） is catalyzed by the cytochrome P450 monooxygenase CYP71A13. Here, we demonstrate that the Ara- bidopsis GH3.5 protein, a multifunctional acetyl-amido synthetase, is involved in camalexin biosynthesis via conjugating indole-3-carboxylic acid （ICA） and cysteine （Cys） and regulating camalexin biosynthesis genes. Camalexin levels were increased in the activation-tagged mutant gh3.5-1D in both Col-0 and cyp71A13-2 mutant backgrounds after pathogen infection. The recombinant GH3.5 protein catalyzed the conjugation of ICA and Cys to form a possible intermediate indole-3-acyl-cysteinate （ICA（Cys）） in vitro. In support of the in vitro reaction, feeding with ICA and Cys increased camalexin levels in Col-0 and gh3.5-1D. Dihydrocamalexic acid （DHCA）, the precursor of camalexin and the substrate for PAD3, was accumulated in gh3.5-1DIpad3-1, suggesting that ICA（Cys） could be an additional precursor of DHCA for camalexin biosynthesis. Furthermore, expression of the major camalexin biosynthesis genes CYP79B2, CYP71A12, CYP71A13 and PAD3 was strongly induced in gh3.5-1D. Our study suggests that GH3.5 is involved in camalexin biosynthesis through direct catalyzation of the formation of ICA（Cys）, and upregulation of the major biosynthetic pathway genes.

Promotion of Arabidopsis immune responses by a rhizosphere fungus via supply of pipecolic acid to plants and selective augment of phytoalexins

The ascomycete insect pathogenic fungi such as Metarhizium species have been demonstrated with the abilities to form the rhizosphere or endophytic relationships with different plants for nutrient exchanges.In this study,after the evident infeasibility of bacterial disease development in the boxed sterile soils,we established a hydroponic system for the gnotobiotic growth of Arabidopsis thaliana with the wild-type and transgenic strain of Metarhizium robertsii.The transgenic fungus could produce a high amount of pipecolic acid(PIP),a pivotal plant-immune-stimulating metabolite.Fungal inoculation experiments showed that M.robertsii could form a non-selective rhizosphere relationship with Arabidopsis.Similar to the PIP uptake by plants after exogenous application,PIP level increased in Col-0 and could be detected in the PIP-non-producing Arabidopsis mutant(ald1)after fungal inoculations,indicating that plants can absorb the PIP produced by fungi.The transgenic fungal strain had a better efficacy than the wild type to defend plants against the bacterial pathogen and aphid attacks.Contrary to ald1,fmo1 plants could not be boosted to resist bacterial infection after treatments.After fungal inoculations,the phytoalexins camalexin and aliphatic glucosinolate were selectively increased in Arabidopsis via both PIP-dependent and-independent ways.This study unveils the potential mechanism of the fungus-mediated beneficial promotion of plant immunity against biological stresses.The data also highlight the added values of M.robertsii to plants beyond the direct suppression of insect pest populations.

Transcriptional Activation and Production of Tryptophan-Derived Secondary Metabolites in Arabidopsis Roots Contributes to the Defense against the Fungal Vascular Pathogen Verticillium Iongisporum

The soil-borne fungal pathogen Verticillium Iongisporum causes vascular disease on Brassicaceae host plants such as oilseed rape. The fungus colonizes the root xylem and moves upwards to the foliage where disease symptoms become visible. Using Arabidopsis as a model for early gene induction, we performed root transcriptome analyses in re- sponse to hyphal growth immediately after spore germination and during penetration of the root cortex, respectively. Infected roots showed a rapid reprogramming of gene expression such as activation of transcription factors, stress-, and defense-related genes. Here, we focused on the highly coordinated gene induction resulting in the production of tryp- tophan-derived secondary metabolites. Previous studies in leaves showed that enzymes encoded by CYP81F2 and PEN2 （PENETRATION2） execute the formation of antifungal indole glucosinolate （IGS） metabolites. In Verticillium-infected roots, we found transcriptional activation of CYP81F2 and the PEN2 homolog PEL 1 （PEN2-LIKE1）, but no increase in antifungal IGS breakdown products. In contrast, indole-3-carboxylic acid （13CA） and the phytoalexin camalexin accumulated in infected roots but only camalexin inhibited Verticillium growth in vitro. Whereas genetic disruption of the individual metabolic pathways leading to either camalexin or CYP81F2-dependent IGS metabolites did not alter Verticillium-induced disease symptoms, a cyp79b2 cyp79b3 mutant impaired in both branches resulted in significantly enhanced susceptibility. Hence, our data provide an insight into root-specific early defenses and suggest tryptophan-derived metabolites as active anti- fungal compounds against a vascular pathogen.

Regulation of Pathogen-Triggered Tryptophan Metabolism in Arabidopsis thaliana by MYB Transcription Factors and Indole Glucosinolate Conversion Products

MYB34, MYB51, and MYB122 transcription factors are known as decisive regulators of indolic glucosinolate （IG） biosynthesis with a strong impact on expression of genes encoding CYP79B2 and CYP79B3 enzymes that redundantly convert tryptophan to indole-3-acetaldoxime （IAOx）. This intermediate represents a branching point for IG biosynthesis, and pathways leading to camalexin and indole-carboxylic acids （ICA）. Here we investigate how these MYBs affect the pathogen-triggered Trp metabolism. Our experiments indicated that these three MYBs affect not only IG production but also constitutive biosynthesis of other IAOx- derived metabolites. Strikingly, the PENETRATION 2 （PEN2）-dependent IG-metabolism products, which are absent in myb34/51/122 and pen2 mutants, were indispensable for full flg22-mediated induction of other IAOx-dedved compounds. However, germ induction and accumulation of ICAs and camalexin upon path- ogen infection was not compromised in myb34/51/122 plants, despite strongly reduced IG levels. Hence, in comparison with cyp79B2/B3, which lacks all IAOx-derived metabolites, we found myb34/51/122 an ideal tool to analyze IG contribution to resistance against the necrotrophic fungal pathogen Plectosphaerella cucumerina. The susceptibility of myb34/51/122 was similar to that of pen2, but much lower than susceptibility of cyp79B2/B3, indicating that MYB34/51/122 contribute to resistance toward P. cucumerina exclu- sively through IG biosynthesis, and that PEN2 is the main leaf myrosinase activating IGs in response to microbial pathogens.

植物对病原微生物的“化学防御”:植保素的生物合成及其分子调控机制

植物可以合成大量的次生代谢物来调控自身对环境的适应性。其中,有一大部分直接参与了植物对病原微生物的防御反应,包括植物在感病前就已经生成和积累的植物毒素,以及感病后才从头合成的抗病小分子次生代谢物—植保素。植保素在植物抵御病原菌,特别是腐生性病原菌的过程中起着非常重要的作用。自从80年前植保素的概念被提出后,大量的植保素从各种植物中被分离和鉴定出来,但它们之中大部分的生物合成和调控机制目前尚不清楚。本文综述了近几年来拟南芥和烟草中植保素的研究进展,特别是它们的生物合成及分子调控机制,并对植保素研究领域存在的问题和今后的研究方向进行了讨论和展望。