Nayyar Shivani, Sharma Bipen Kumar, Kaur Ajinder, Kalia Anu, Sanghera Gulzar Singh, Thind Karanjit Singh, Yadav Inderjit Singh, Sandhu Jagdeep Singh
School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India.
Punjab Agricultural University, Regional Research Station, Kapurthala, India.
PLoS One. 2017 Jun 28;12(6):e0179723. doi: 10.1371/journal.pone.0179723. eCollection 2017.
Sugarcane (Saccharum spp.) is a commercially important crop, vulnerable to fungal disease red rot caused by Colletotrichum falcatum Went. The pathogen attacks sucrose accumulating parenchyma cells of cane stalk leading to severe losses in cane yield and sugar recovery. We report development of red rot resistant transgenic sugarcane through expression of β-1,3-glucanase gene from Trichoderma spp. The transgene integration and its expression were confirmed by quantitative reverse transcription-PCR in first clonal generation raised from T0 plants revealing up to 4.4-fold higher expression, in comparison to non-transgenic sugarcane. Bioassay of transgenic plants with two virulent C. falcatum pathotypes, Cf 08 and Cf 09 causing red rot disease demonstrated that some plants were resistant to Cf 08 and moderately resistant to Cf 09. The electron micrographs of sucrose storing stalk parenchyma cells from these plants displayed characteristic sucrose-filled cells inhibiting Cf 08 hyphae and lysis of Cf 09 hyphae; in contrast, the cells of susceptible plants were sucrose depleted and prone to both the pathotypes. The transgene expression was up-regulated (up to 2.0-fold in leaves and 5.0-fold in roots) after infection, as compared to before infection in resistant plants. The transgene was successfully transmitted to second clonal generation raised from resistant transgenic plants. β-1,3-glucanase protein structural model revealed that active sites Glutamate 628 and Aspartate 569 of the catalytic domain acted as proton donor and nucleophile having role in cleaving β-1,3-glycosidic bonds and pathogen hyphal lysis.
甘蔗（甘蔗属）是一种具有重要商业价值的作物，易受由镰孢炭疽菌（Colletotrichum falcatum Went）引起的真菌病害——赤腐病的侵害。该病原菌侵袭甘蔗茎中积累蔗糖的薄壁细胞，导致甘蔗产量和糖分回收率严重下降。我们报道了通过表达木霉属的β-1,3-葡聚糖酶基因培育抗赤腐病转基因甘蔗的过程。通过定量逆转录聚合酶链反应在从T0植株培育的第一代克隆植株中证实了转基因的整合及其表达，与非转基因甘蔗相比，其表达量提高了4.4倍。用两种引起赤腐病的强毒力镰孢炭疽菌致病型Cf 08和Cf 09对转基因植株进行生物测定，结果表明一些植株对Cf 08具有抗性，对Cf 09具有中度抗性。这些植株中储存蔗糖的茎薄壁细胞的电子显微镜照片显示，细胞充满特征性的蔗糖，抑制了Cf 08菌丝，导致Cf 09菌丝溶解；相比之下，感病植株的细胞蔗糖耗尽，对两种致病型都易感。与抗性植株感染前相比，感染后转基因表达上调（叶片中高达2.0倍，根中高达5.0倍）。转基因成功地传递到了从抗性转基因植株培育的第二代克隆植株中。β-1,3-葡聚糖酶蛋白结构模型显示，催化结构域的活性位点谷氨酸628和天冬氨酸569作为质子供体和亲核试剂，在裂解β-1,3-糖苷键和病原菌菌丝溶解中发挥作用。
