National Key Laboratory of Crop Genetics and Germplasm Enhancement, Cotton Research Institute, Nanjing Agricultural University, Nanjing 210095, China.
J Genet Genomics. 2013 Aug 20;40(8):421-31. doi: 10.1016/j.jgg.2013.04.006. Epub 2013 May 9.
Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10(6) conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V. dahliae.
自噬是真核生物中一种广泛保守的细胞内过程，用于降解和回收蛋白质、细胞器和细胞质，现在它作为许多植物病原真菌叶片感染的一个重要过程而出现。然而，自噬在土壤真菌生理和感染生物学中的作用还知之甚少。在这里，我们报告了建立一种农杆菌介导的转化（ATMT）系统及其在通过靶向基因替换和互补来研究两个自噬基因 VdATG8 和 VdATG12 中的应用。利用新型二元载体 pCOM 对棉花感染的黄萎病菌 Vd8 进行转化，每 1×10(6)个分生孢子产生 384 个遗传霉素抗性转化体。缺乏 VdATG8 或 VdATG12 的 V. dahliae 突变体表现出分生孢子减少和气生菌丝产生受损。与野生型和基因互补菌株相比，用 VdATG8 或 VdATG12 基因缺失突变体接种拟南芥植物时，发病进程略有延迟。令人惊讶的是，用未受损的根进行体外接种时，根入侵能力在基因缺失突变体中不受影响。这些结果表明，自噬对于气生菌丝的发育和植物定殖是必要的，但对于黄萎病菌的根侵染则不是必需的。
