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与桦树花粉相关的食物过敏类型之一。大约24%的食物过敏患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的白桦(Betula Verrucosa)花粉抗原Bet v1与同源食物蛋白之间的交叉反应IgE表位引起的。BET v1和胡萝卜(Daucus Carota)主要变应原DAUc1属于致病相关蛋白10(PR10)家族。PR10是一种普遍存在的小分子植物蛋白，受微生物和非生物胁迫诱导，对包括胡萝卜在内的许多水果和蔬菜的过敏性有不利影响。在DFG-PAK132的第二个资助期(请注意，这是AG Kogel的第一个资助期)，我们利用RNA干扰技术(RNAi)沉默了DAU c 1亚型DAU c 1.01和DAU c 1.02，从而创造了低过敏性胡萝卜植株。此外，我们还鉴定了第三个新的亚型DAUC1.03，其致敏潜力由项目合作伙伴Mahler和Vieths显示。通过皮肤点刺试验，DAU c 1.01-和DAU c 1.02沉默的胡萝卜植株的致敏效力的降低足以导致胡萝卜过敏患者的致敏反应性降低。对沉默植物的抗病性评估表明，与野生型胡萝卜相比，沉默植物对病原菌Alternaria dauci、Alternaria Radina和Botrytis cinerea的敏感性没有差异。虽然DAU c1亚型明显导致过敏，但对它们在胡萝卜发育和抗病中的作用方式知之甚少。基于DFG-PAK132前一资助阶段的结果，拟议的研究计划旨在进一步鉴定转基因低致敏性胡萝卜，这些胡萝卜将通过多次沉默(敲除)DAU c1亚型产生。为了获得双重和三重DAUc1沉默的胡萝卜植株，将使用RNAi的突变方法以及一种新的使用TALE核酸酶(TALES：转录激活因子样效应器)的靶向基因敲除技术。将对沉默/敲除的DAUc1植物进行表征，并测试其对常见胡萝卜病原体的抗性反应的变化。此外，选定的品系将提供给我们的项目合作伙伴，用于评估(I)蛋白质组成的潜在补偿性变化，以及(Ii)通过皮肤点刺试验、口腔刺激和生物测定的过敏性效力。我们还将通过对项目合作伙伴Sonnewald小组培育的转基因低过敏性番茄植株进行病理性鉴定，为DFG-PAK132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.