Jashni Mansoor Karimi, Dols Ivo H M, Iida Yuichiro, Boeren Sjef, Beenen Henriek G, Mehrabi Rahim, Collemare Jérôme, de Wit Pierre J G M
1 Laboratory of Phytopathology, Wageningen University and Research Centre, 6708 PB, Wageningen, The Netherlands;
2 Department of Plant Pathology, Tarbiat Modares University, 14115-336, Tehran, Iran;
Mol Plant Microbe Interact. 2015 Sep;28(9):996-1008. doi: 10.1094/MPMI-04-15-0074-R. Epub 2015 Aug 10.
As part of their defense strategy against fungal pathogens, plants secrete chitinases that degrade chitin, the major structural component of fungal cell walls. Some fungi are not sensitive to plant chitinases because they secrete chitin-binding effector proteins that protect their cell wall against these enzymes. However, it is not known how fungal pathogens that lack chitin-binding effectors overcome this plant defense barrier. Here, we investigated the ability of fungal tomato pathogens to cleave chitin-binding domain (CBD)-containing chitinases and its effect on fungal virulence. Four tomato CBD chitinases were produced in Pichia pastoris and were incubated with secreted proteins isolated from seven fungal tomato pathogens. Of these, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae, and Botrytis cinerea were able to cleave the extracellular tomato chitinases SlChi1 and SlChi13. Cleavage by F. oxysporum removed the CBD from the N-terminus, shown by mass spectrometry, and significantly reduced the chitinase and antifungal activity of both chitinases. Both secreted metalloprotease FoMep1 and serine protease FoSep1 were responsible for this cleavage. Double deletion mutants of FoMep1 and FoSep1 of F. oxysporum lacked chitinase cleavage activity on SlChi1 and SlChi13 and showed reduced virulence on tomato. These results demonstrate the importance of plant chitinase cleavage in fungal virulence.
作为其抵御真菌病原体防御策略的一部分，植物分泌几丁质酶来降解几丁质，几丁质是真菌细胞壁的主要结构成分。一些真菌对植物几丁质酶不敏感，因为它们分泌几丁质结合效应蛋白来保护其细胞壁免受这些酶的作用。然而，尚不清楚缺乏几丁质结合效应蛋白的真菌病原体如何克服这种植物防御屏障。在这里，我们研究了番茄真菌病原体切割含几丁质结合结构域（CBD）的几丁质酶的能力及其对真菌毒力的影响。在毕赤酵母中产生了四种番茄CBD几丁质酶，并与从七种番茄真菌病原体中分离的分泌蛋白一起孵育。其中，尖孢镰刀菌番茄专化型、大丽轮枝菌和灰葡萄孢能够切割细胞外番茄几丁质酶SlChi1和SlChi13。质谱分析表明，尖孢镰刀菌的切割作用从N端去除了CBD，并显著降低了两种几丁质酶的几丁质酶活性和抗真菌活性。分泌的金属蛋白酶FoMep1和丝氨酸蛋白酶FoSep1都参与了这种切割作用。尖孢镰刀菌的FoMep1和FoSep1双缺失突变体对SlChi1和SlChi13缺乏几丁质酶切割活性，并且在番茄上的毒力降低。这些结果证明了植物几丁质酶切割在真菌毒力中的重要性。
