Song Na, Dai Qingqing, Zhu Baitao, Wu Yuxing, Xu Ming, Voegele Ralf Thomas, Gao Xiaoning, Kang Zhensheng, Huang Lili
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, People's Republic of China.
Fachgebiet Phytopathologie, Institut für Phytomedizin, Universitӓt Hohenheim, Stuttgart, Germany.
PLoS One. 2017 Mar 7;12(3):e0173141. doi: 10.1371/journal.pone.0173141. eCollection 2017.
In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. 'Fuji' leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes-important virulence factors in V. mali-are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.
在真菌中，异源三聚体鸟嘌呤核苷酸结合蛋白（G蛋白）是信号转导途径的关键元件，其控制生长、无性和有性发育以及毒力。在本研究中，我们从苹果腐烂病的病原菌苹果黑腐皮壳菌中鉴定出两个编码异源三聚体G蛋白α亚基的基因，命名为Gvm2和Gvm3。Gvm2和Gvm3突变体的特征表明，Gvm3可能是营养生长的关键调节因子。相应基因的缺失导致生长速率降低20%。此外，Gvm2和Gvm3似乎参与无性繁殖，并且突变体对氧化应激和细胞膜应激高度敏感。有趣的是，这两个G蛋白α亚基很可能都参与了苹果黑腐皮壳菌的毒力。在用苹果品种‘富士’叶片和嫩枝进行的感染试验中，缺失突变体△Gvm2或△Gvm3引起的病斑大小显著减小。此外，许多编码水解酶（苹果黑腐皮壳菌中重要的毒力因子）的基因在这些缺失突变体中的表达水平较低。我们的结果表明，Gvm2和Gvm3可能通过调节细胞壁降解酶的表达在毒力中发挥重要作用。进一步分析了△Gvm2和△Gvm3突变体对cAMP/PKA途径中基因转录水平的影响。编码腺苷酸环化酶VmAC、蛋白激酶A（PKA）调节亚基VmPKR和PKA催化亚基VmPKA1的基因在两个突变体中的表达均下调。进一步分析表明，△Gvm3突变体中细胞内cAMP水平和PKA活性下调，但△Gvm2突变体中基本不变。总体而言，我们的研究结果表明，Gvm2和Gvm3在调节苹果黑腐皮壳菌的营养生长、无性发育和毒力方面发挥着多种作用。
