马铃薯Y病毒HC-Pro第182位赖氨酸残基参与引起烟草叶脉坏死

 马铃薯Y病毒(potato virus Y,PVY)是侵染烟草的最重要病毒之一。不同PVY株系侵染烟草可引起不同症状,有些PVY株系可引起烟草叶脉坏死,严重影响烟草的产量和品质。PVY A12分离物属于NTN-NW株系,但侵染珊西烟(Nicotiana tabacum cv. Xanthi)不能引起叶脉坏死。分析发现,PVY分离物A12 HC-Pro第182和245位的氨基酸均为精氨酸(R),而能引起叶脉坏死的其他NTN-NW分离物HC-Pro的这2个位点均为赖氨酸(K)。PVY坏死株系N605的HC-Pro第182位和245位氨基酸也均为K。本研究通过定点突变,将N605侵染性克隆PVY^N605-GFP HC-Pro第245位残基K突变为R,突变体仍然能够引起叶脉坏死,而将其HC-Pro第182位残基K突变为R,突变体不能引起叶脉坏死。Western blot检测发现,2个突变体与野生型病毒CP蛋白在珊西烟中的表达水平没有明显差异。沉默抑制实验结果显示,2个突变体和野生型的HC-Pro抑制RNA沉默能力没有发生变化。初步确定PVY^N605-GFP HC-Pro第182残基K是引起叶脉坏死的关键氨基酸,推测PVY A12分离物不能引起烟草叶脉坏死的原因是其HC-Pro第182残基R引起的。

The residue lysine at position 182 in HC-Pro of potato virus Y is involved in the veinal necrosis symptom in Nicotiana tabacum

Potato virus Y is one of the major viruses infecting tobacco plant and different strains induce different symptom in tobacco. Some PVY isolates induce veinal necrosis in Nicotiana tabacum and severely affect the yield and quality of tobacco leaves. The PVY isolate A12 belongs to the NTN-NW strain, and induces symptom of mosaic, instead of veinal necrosis in Nicotiana tabacum cv. Xanthi. The other NTN-NW isolates that induce veinal necrosis in Nicotiana tabacum have the amino acid residue lysine (K) at both positions 182 and 245, while A12 has the residue arginine (R) at both positions. The corresponding amino acid residues at positions 182 and 245 are also K in the HC-Pro of PVY necrosis isolate N605. Mutation was introduced to infectious clone PVY^N605-GFP via site-directed mutagenesis. The inoculation results showed that the mutant with substitution of R for K at position 182 in HC-Pro could not induce veinal necrosis in N. tabacum cv. Xanthi. Western blot results showed that there was no significant difference in the CP accumulation levels between the wild type and mutant PVY. The RNA silencing suppression assay showed that there was no significant change between wild type and mutant HC-Pro. Therefore, we concluded that the residue K at position 182 of HC-Pro was an critical amino acid responsible for tobacco veinal necrosis, and presumed that the residue R at position 182 of HC-Pro was accountable for the failure of A12 to induce tobacco veinal necrosis.