Prothction of disease-resistant plants by cell engineering using host-specific toxins of pathogens

已知某些真菌病原体产生宿主特有的毒素(HST)，这些毒素决定了它们的寄主范围，并有助于它们的毒力或致病性。近年来，利用HSTs从感病植物中选育抗病基因的技术比较流行。在许多植物中，通过组织或细胞在培养过程中的生长产生的体细胞突变释放出新的抗病性状，并利用HSTs进行体外筛选。我们发现，油菜链格孢菌(Alternaria Brassicola)、黄瓜致病型和番茄致病型黄瓜棒孢霉(Corynespora Cassiicola)和泡囊茎瘤菌(Stemphlium Vesicarium)分别产生新的HSTs，命名为AB-、CCC-、CCT-和SV-毒素。AB-毒素是一种蛋白质，分子量为35 kDa，CCT-毒素是一种环状多肽，分子量为2780。CCT-毒素用于番茄抗病育种。以番茄下胚轴为外植体，诱导出再生频率较高的绿斑愈伤组织，并转移到含有CCT-毒素的MS培养基上。虽然几乎所有的愈伤组织都变成了棕色，但有些愈伤组织在孵化3周后仍然存活下来。然而，耐毒素的愈伤组织失去了再生能力。另一方面，番茄赤霉病病型产生一种AAL毒素(HST)。最近，从黑酵母Spinifera中克隆到了代谢AAL-毒素的ESP1。因此，尝试利用转ESP1基因番茄的AAL毒素脱毒培育抗病番茄。利用含有ESP1双元载体的根癌农杆菌对番茄进行了ESP1的转化。然而，目前还没有获得转化子。

Certain fungal pathogens are known to produce host-specific toxins (HSTs) that determine their host range and contribute to their virulence or pathogenicity. Recently, technology for breeding disease-resistant genotypes from susceptible plants by using HSTs is relatively prevalent. In many plants, somatic mutations generated through the growth of tissues or cells in culture release novel disease resistance characters, and HSTs are used to select in vitro for such characters.We found that Alternaria brassicicola, cucumber pathotype and tomato pathotype of Corynespora cassiicola, and Stemphylium vesicarium produce new HSTs named AB-, CCC-, CCT- and SV-toxins, respectively. AB-toxin was a protein and estimated to be 35 kDa, and CCT-toxin was a cyclic peptide with molecular weight of 2,780.CCT-toxin of C.cassiicola tomato pathotype was used for production of disease-resistant tomato. Tomato calli with green spots which have a high regeneration frequency were induced from hypocotyls and transferred to MS medium containing CCT-toxin. Although almost all calli became brown, some calli continued to survive even after incubation for 3 weeks. However, the toxin-tolerant calli lost ability to regenerate. On the other hand, A.alternata tomato pathotype produces a HST (AAL-toxin). Recently, the ESP1 which metabolizes AAL-toxin was cloned from black yeast Exophilia spinifera. Therefore, production of disease-resistant tomato by detoxification of AAL-toxin in ESP1-transgenic tomato was attempted. Transformation of ESP1 in tomato was performed by Agrobacterium tumefaciens containing binary vector with ESP1. However, transformants have not yet been obtained.