Barad Shiri, Espeso Eduardo A, Sherman Amir, Prusky Dov
Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan, 50250, Israel.
Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, 76100, Israel.
Mol Plant Pathol. 2016 Jun;17(5):727-40. doi: 10.1111/mpp.12327. Epub 2015 Dec 3.
Penicillium expansum, the causal agent of blue mould rot, causes severe post-harvest fruit maceration simultaneously with the secretion of d-gluconic acid (GLA) and the mycotoxin patulin in colonized tissue. The factor(s) inducing patulin biosynthesis during colonization of the host acidic environment is unclear. During the colonization of apple fruit in vivo and growth in culture, P. expansum secretes pH-modulating GLA and ammonia. Although patulin and its possible opportunistic precursor GLA accumulate together during fungal development, ammonia is detected on the colonized tissue's leading edge and after extended culture, close to patulin accumulation. Here, we demonstrate ammonia-induced transcript activation of the global pH modulator PacC and patulin accumulation in the presence of GLA by: (i) direct exogenous treatment of P. expansum growing on solid medium; (ii) direct exogenous treatment on colonized apple tissue; (iii) growth under self-ammonia production conditions with limited carbon; and (iv) analysis of the transcriptional response to ammonia of the patulin biosynthesis cluster. Ammonia induced patulin accumulation concurrently with the transcript activation of pacC and patulin biosynthesis cluster genes, indicating the regulatory effect of ammonia on pacC transcript expression under acidic conditions. Electrophoretic mobility shift assays using P. expansum PacC and antibodies to the different cleaved proteins showed that PacC is not protected against proteolytic signalling at pH 4.5 relative to pH 7.0, but NH4 addition did not further enhance its proteolytic cleavage. Ammonia enhanced the activation of palF transcript in the Pal pathway under acidic conditions. Ammonia accumulation in the host environment by the pathogen under acidic pH may be a regulatory cue for pacC activation, towards the accumulation of secondary metabolites, such as patulin.
扩展青霉是青霉腐烂病的病原菌，在侵染组织中会导致严重的采后果实浸软，同时分泌d - 葡萄糖酸（GLA）和霉菌毒素棒曲霉素。在宿主酸性环境定殖过程中诱导棒曲霉素生物合成的因素尚不清楚。在苹果果实体内定殖和体外培养生长过程中，扩展青霉会分泌调节pH值的GLA和氨。尽管在真菌发育过程中棒曲霉素及其可能的机会性前体GLA会一起积累，但在侵染组织的前沿以及延长培养后靠近棒曲霉素积累处可检测到氨。在此，我们通过以下方式证明了氨在GLA存在的情况下诱导全局pH调节剂PacC的转录激活以及棒曲霉素积累：（i）直接对外源生长在固体培养基上的扩展青霉进行处理；（ii）直接对外源侵染的苹果组织进行处理；（iii）在有限碳源的自氨产生条件下生长；（iv）分析棒曲霉素生物合成簇对氨的转录反应。氨诱导棒曲霉素积累的同时伴随着pacC和棒曲霉素生物合成簇基因的转录激活，表明在酸性条件下氨对pacC转录表达具有调节作用。使用扩展青霉PacC和针对不同切割蛋白的抗体进行的电泳迁移率变动分析表明，相对于pH 7.0，PacC在pH 4.5时不能免受蛋白水解信号的影响，但添加NH4并未进一步增强其蛋白水解切割。在酸性条件下，氨增强了Pal途径中palF转录本的激活。病原菌在酸性pH值下在宿主环境中积累氨可能是pacC激活的调节信号，促使次生代谢产物如棒曲霉素的积累。
