Mechanistic analysis of the defence status of low-allergenic carrot and tomato plants

胡萝卜过敏是中欧最常见的桦树花粉相关食物过敏类型之一。大约24%的食物过敏受试者在摄入胡萝卜后出现过敏症状。对胡萝卜的不良反应是由于主要白色桦树（Betula verrucosa）花粉抗原Bet v 1和同源食物蛋白之间的交叉反应性IgE表位引起的。Bet v 1和胡萝卜（Daucus carota）的主要过敏原Dau c 1属于发病相关蛋白10（PR 10）家族。PR 10是一种普遍存在的小分子植物蛋白，由微生物病原体和非生物胁迫诱导，并对许多水果和蔬菜（包括胡萝卜）的致敏性产生不利影响。在DFG-PAK 132的第二个资助期（请注意这是AG Kogel的第一个资助期），我们通过使用RNA干扰技术（RNAi）沉默Dau c 1同种型Dau c 1.01和Dau c 1.02来创建低变应原性胡萝卜植物。此外，我们确定了第三种新的同种型Dau c 1.03，其致敏潜力已由项目合作伙伴Mahler和Vieths证明。Dau c 1.01-和Dau c 1.02沉默胡萝卜植物中变应原效力的降低足以引起胡萝卜过敏患者的变应原反应性降低，如皮肤点刺试验所确定的。评估沉默植物的抗病性表明，与野生型carrots.While Dau c 1亚型清楚地有助于过敏，很少有人知道他们的行动方式在胡萝卜的发展和抗病性的植物病原体，胡萝卜链格孢，根链格孢，灰葡萄孢菌的易感性没有差异。基于DFG-PAK 132上一个资助期获得的结果，拟议的研究计划旨在进一步表征转基因低过敏性胡萝卜，这些胡萝卜将通过Dau c 1亚型的多重沉默（敲除）产生。为了获得双重和三重Dau c 1沉默的胡萝卜植物，将采用通过RNAi的突变方法以及使用TALE核酸酶（TALE：转录激活因子样效应物）的新型靶向基因敲除技术。Dau c1沉默/敲除植物将被表征并测试对常见胡萝卜病原体的抗性应答的改变。此外，将向我们的项目合作伙伴提供选定的细胞系，以评估（i）蛋白质组成的潜在代偿性变化，以及（ii）通过皮肤点刺试验、口服激发和生物测定的致敏效力。我们还将通过对项目合作伙伴Sonnewald的小组中产生的转基因低过敏性番茄植物进行病理分型，为DFG-PAK 132做出贡献。

Carrot allergy is one of the most common types of birch pollen-related food allergy in central Europe. Approximately 24% of food allergic subjects suffer from allergic symptoms after ingestion of carrot. Adverse reactions to carrots are evoked due to cross-reactive IgE epitopes between the major white birch (Betula verrucosa) pollen antigen Bet v 1 and homologous food proteins. Bet v 1 and the major carrot (Daucus carota) allergen Dau c 1 belong to the pathogenesis-related protein-10 (PR10) family. PR10 is a ubiquitous small plant protein induced both by microbial pathogens and abiotic stress, and adversely contributes to the allergenic potency of many fruits and vegetables, including carrot. Two highly similar genes encoding PR10 isoforms have been isolated and designated as carrot allergens Dau c 1.01 and Dau c 1.02.During the second funding period of DFG-PAK132 (please note this is the first for AG Kogel) we created hypoallergenic carrot plants via silencing the Dau c 1 isoforms Dau c 1.01 and Dau c 1.02 using the RNA interference technology (RNAi). Furthermore, we identified a third novel isoform Dau c 1.03 whose allergenic potential was shown by project partner Mahler and Vieths. The decrease of the allergenic potency in Dau c 1.01- and Dau c 1.02 silenced carrot plants was sufficient to cause a reduced allergenic reactivity in patients with carrot allergy, as determined with skin prick tests. Assessment of silenced plants for disease resistance showed no difference in susceptibility to the plant pathogens Alternaria dauci, Alternaria radicina, and Botrytis cinerea, as compared with wild type carrots.While Dau c 1 isoforms clearly contribute to allergies, little is known about their mode of action in carrot development and disease resistance. Based on the results obtained from the previous funding period of DFG-PAK132, the proposed research program aims to further characterize transgenic hypoallergenic carrots that will be created by multiple silencing (knockout) of the Dau c 1 isoforms. To obtain double and triple Dau c 1 silenced carrot plants, mutational approaches by RNAi as well as a novel targeted gene knock-out technique using TALE nucleases (TALEs: transcription activator-like effectors) will be employed. Dau c 1-silenced/knockout plants will be characterized and tested for altered resistance responses to common carrot pathogens. Furthermore, selected lines will be provided to our project partner for the assessment of (i) potential compensatory changes in the protein composition, and (ii) allergenic potency by skin prick tests, oral provocation, and bioassays. We will also contribute to DFG-PAK132 by pathotyping transgenic hypoallergenic tomato plants generated in the group of the project partner Sonnewald.