Mukherjee Mala, Mukherjee Prasun K, Kale Sharad P
Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India.
Microbiology (Reading). 2007 Jun;153(Pt 6):1734-1742. doi: 10.1099/mic.0.2007/005702-0.
An adenylate-cyclase-encoding gene, tac1, of Trichoderma virens, a soil fungus used in the biocontrol of plant pathogens, has been cloned and sequenced. The tac1 ORF spanned 7032 bp, encoding a protein of 2153 aa, which shared an identity of 65 % with the adenylate cyclase of Colletotrichum lagenarium. Deletion of tac1, through double-crossover homologous recombination, lowered the intracellular cAMP levels to below the detection limit. The mutants showed only 5-6 % of the wild-type growth rate on agar, but grew normally in shake culture. The mutants did not sporulate in darkness, and the spores failed to germinate in water. In the confrontation assay, the mutants did not overgrow the test plant pathogens Sclerotium rolfsii, Rhizoctonia solani and Pythium sp. Against Pythium sp., the mutants produced a clear zone of inhibition in the confrontation assay. HPLC analysis and bioassay showed reduced secondary metabolite production in the mutants. Using suppression subtractive hybridization (SSH), the genes that were underexpressed in the mutants were identified. Based on an array of 53 SSH library clones, 11 clones were identified as strongly downregulated in the Deltatac1 mutants; of these 11 clones, nine sequences were homologous to secondary metabolism-related gene sequences. Therefore, cAMP signalling positively regulates secondary metabolism in T. virens. This is believed to be the first direct genetic study on the role of cAMP signalling in a Trichoderma sp. Tac1 is also believed to be the first regulatory protein to be identified in T. virens that is involved in growth, germination, mycoparasitism and secondary metabolism.
哈茨木霉是一种用于生物防治植物病原体的土壤真菌，其腺苷酸环化酶编码基因tac1已被克隆和测序。tac1开放阅读框跨度为7032 bp，编码一个2153个氨基酸的蛋白质，该蛋白质与葫芦炭疽菌的腺苷酸环化酶具有65%的同一性。通过双交换同源重组缺失tac1，可使细胞内cAMP水平降至检测限以下。突变体在琼脂上的生长速率仅为野生型的5-6%，但在摇瓶培养中生长正常。突变体在黑暗中不产孢，孢子在水中不能萌发。在对峙试验中，突变体不会过度生长于测试植物病原体立枯丝核菌、茄丝核菌和腐霉菌。针对腐霉菌，突变体在对峙试验中产生了明显的抑制圈。高效液相色谱分析和生物测定表明，突变体中次生代谢产物的产量降低。利用抑制性消减杂交（SSH）技术，鉴定了突变体中表达下调的基因。基于53个SSH文库克隆的阵列，11个克隆被鉴定为在Δtac1突变体中强烈下调；在这11个克隆中，9个序列与次生代谢相关基因序列同源。因此，cAMP信号通路正向调节哈茨木霉的次生代谢。这被认为是关于cAMP信号通路在木霉属中作用的首次直接遗传学研究。Tac1也被认为是在哈茨木霉中鉴定出的第一个参与生长、萌发、真菌寄生和次生代谢的调节蛋白。
