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

胡萝卜过敏是中欧桦树花粉相关食物过敏最常见的类型之一。大约24%的食物过敏受试者在食用胡萝卜后出现过敏症状。胡萝卜的不良反应是由于白桦花粉抗原betv1与同源食物蛋白之间的IgE表位交叉反应引起的。betv1和胡萝卜（Daucus carota）主要过敏原dac1属于致病相关蛋白-10 （PR10）家族。PR10是一种普遍存在的植物小蛋白，可由微生物病原体和非生物胁迫诱导，并对包括胡萝卜在内的许多水果和蔬菜的致敏效力起不利作用。两个高度相似的编码PR10亚型的基因被分离出来，并被命名为胡萝卜过敏原Dau c 1.01和Dau c 1.02。在DFG-PAK132的第二个资助期（请注意，这是AG Kogel的第一个资助期），我们通过RNA干扰技术（RNAi）沉默Dau c1亚型Dau c 1.01和Dau c 1.02，创造了低致敏性的胡萝卜植株。此外，我们发现了第三种新的异构体Dau c 1.03，其致敏潜力由项目合作伙伴Mahler和Vieths证实。通过皮肤点刺试验确定，Dau c 1.01-和Dau c 1.02沉默胡萝卜植株的致敏效力降低，足以导致胡萝卜过敏患者的致敏反应性降低。对沉默植株抗病性的评估显示，与野生型胡萝卜相比，沉默植株对植物病原菌红锈菌（Alternaria dauci）、根茎锈菌（Alternaria radicina）和灰霉病菌（Botrytis cinerea）的易感性没有差异。虽然Dau c1异构体明显与过敏有关，但它们在胡萝卜发育和抗病性中的作用模式却知之甚少。基于DFG-PAK132之前资助期获得的结果，拟议的研究计划旨在进一步表征转基因低过敏性胡萝卜，这些胡萝卜将通过多次沉默（敲除）Dau c1亚型而产生。为了获得双重和三重Dau c1沉默的胡萝卜植株，将采用RNAi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 c 1沉默/敲除植物将被鉴定并测试其对常见胡萝卜病原体的抗性反应。此外，选定的品系将提供给我们的项目合作伙伴，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性。我们还将通过对项目合作伙伴Sonnewald小组产生的转基因低过敏性番茄植株进行病理分型，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.