Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, the Republic of Korea.
Plant Immunity Research Center, Seoul National University, Seoul 08826, the Republic of Korea.
Microbiol Res. 2024 Aug;285:127743. doi: 10.1016/j.micres.2024.127743. Epub 2024 May 7.
Clavibacter michiganensis is a Gram-positive bacterium that causes diverse disease symptoms in tomatoes and Nicotiana benthamiana, a surrogate host plant, including canker, blister lesions, and wilting. Previously, we reported that C. michiganensis also causes necrosis in N. benthamiana leaves. Here, to identify novel virulence genes of C. michiganensis required for necrosis development in N. benthamiana leaves, we screened 1,862 transposon-inserted mutants and identified a mutant strain that exhibited weak and delayed necrosis, whereas there was no discernible difference in blister lesions, canker, or wilting symptoms. Notably, this mutant caused canker similar to that of the wild-type strain, but caused mild wilting in tomato. This mutant carried a transposon in a chromosomal gene, called Clavibactervirulence gene A1 (cviA1). CviA1 encodes a 180-amino acid protein with a signal peptide (SP) at the N-terminus and two putative transmembrane domains (TMs) at the C-terminus. Interestingly, deletion of the SP or the C-terminus, including the two putative TMs, in CviA1 failed to restore full necrosis in the mutant, highlighting the importance of protein secretion and the putative TMs for necrosis. A paralog of cviA1, cviA2 is located on the large plasmid pCM2 of C. michiganensis. Despite its high similarity to cviA1, the introduction of cviA2 into the cviA1 mutant strain did not restore virulence. Similarly, the introduction of cviA1 into the Clavibacter capsici type strain PF008, which initially lacks cviA1, did not enhance necrosis symptoms. These results reveals that the chromosomal cviA1 gene in C. michiganensis plays an important role in necrosis development in N. benthamiana leaves.
密歇根棒杆菌是一种革兰氏阳性细菌，可导致番茄和替代宿主植物烟草原生质体出现多种疾病症状，包括溃疡、疱斑和萎蔫。此前，我们报道称，密歇根棒杆菌还可导致烟草原生质体叶片坏死。在此，为鉴定参与烟草原生质体叶片坏死的密歇根棒杆菌新的毒性基因，我们筛选了 1862 个转座子插入突变体，并鉴定出一个表现出弱且延迟坏死的突变株，而疱斑、溃疡或萎蔫症状则无明显差异。值得注意的是，该突变株引起的溃疡类似于野生型菌株，但在番茄中引起轻度萎蔫。该突变株的一个染色体基因携带一个转座子，称为棒杆菌毒性基因 A1（cviA1）。CviA1 编码一个 180 个氨基酸的蛋白质，N 端有一个信号肽（SP），C 端有两个假定的跨膜结构域（TMs）。有趣的是，CviA1 的 SP 或 C 端（包括两个假定的 TMs）缺失均未能使突变株完全恢复坏死，这突出了蛋白分泌和假定 TMs 对坏死的重要性。CviA1 的一个旁系同源物 cviA2 位于密歇根棒杆菌的大型质粒 pCM2 上。尽管 cviA2 与 cviA1 高度相似，但将 cviA2 导入 cviA1 突变株中并不能恢复毒力。同样，将 cviA1 导入最初缺乏 cviA1 的辣椒棒杆菌 PF008 型菌株中也不能增强坏死症状。这些结果表明，密歇根棒杆菌染色体上的 cviA1 基因在烟草原生质体叶片坏死的发展中起着重要作用。
