Sun Hao, Del Toro Francisco, Makki Mongia, Tenllado Francisco, Canto Tomas
Department of Microbial and Plant Biotechnology, Margarita Salas Center for Biological Research, Spanish National Research Council (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
Laboratory of Molecular Genetics, Immunology and Biotechnology, Faculty of Sciences, University of Tunis El Manar, Manar II, Tunis 2092, Tunisia.
Plants (Basel). 2022 Aug 30;11(17):2262. doi: 10.3390/plants11172262.
A viral chimera in which the bi-cistron of a plum pox virus construct (PPV-GFP) was replaced by that of potato virus Y (PVY) spread slowly systemically in plants and accumulated to levels that were 5-10% those of parental PPV-GFP. We tested whether consecutive mechanical passages could increase its virulence, and found that after several passages, chimera titers rose and symptoms increased. We sequenced over half the genome of passaged chimera lineages infecting two plants. The regions sequenced were ; ; , because of being potential sites for mutations/deletions leading to adaptation. We found few substitutions, all non-synonymous: two in one chimera (nt 2053 , and 5733 ), and three in the other (2359 , 5729 , 9466 ). substitutions 2053 AU(Ile)→AU(Thr), and 2359 CG(Leu)→CG(Arg) occurred at positions where single nucleotide polymorphisms were observed in NGS libraries of sRNA reads from agroinfiltrated plants (generation 1). Remarkably, position 2053 was the only one in the sequenced protein-encoding genome in which polymorphisms were common to the four libraries, suggesting that selective pressure existed to alter that specific nucleotide, previous to any passage. Mutations 5729 and 5733 in the by contrast did not correlate with polymorphisms in generation 1 libraries. Reverse genetics showed that substitution 2053 alone increased several-fold viral local accumulation, speed of systemic spread, and systemic titers.
一种病毒嵌合体，其中李痘病毒构建体（PPV-GFP）的双顺反子被马铃薯Y病毒（PVY）的双顺反子所取代，该嵌合体在植物中系统传播缓慢，积累水平仅为亲本PPV-GFP的5%-10%。我们测试了连续机械传代是否能增加其毒力，发现经过几次传代后，嵌合体滴度上升，症状加重。我们对感染两株植物的传代嵌合体谱系的一半以上基因组进行了测序。测序区域为 ； ； ，因为这些区域是导致适应性突变/缺失的潜在位点。我们发现了很少的替换，均为非同义替换：一个嵌合体中有两个（核苷酸位置2053 ，以及5733 ），另一个中有三个（2359 ，5729 ，9466 ）。替换2053处的AU（异亮氨酸）→AU（苏氨酸），以及2359处的CG（亮氨酸）→CG（精氨酸）发生在从农杆菌浸润植物（第1代）的小RNA读数的NGS文库中观察到单核苷酸多态性的位置。值得注意的是，位置2053是测序的蛋白质编码基因组中唯一在四个文库中都存在多态性的位置，这表明在任何传代之前就存在改变该特定核苷酸的选择压力。相比之下， 中的5729和5733突变与第1代文库中的多态性无关。反向遗传学表明，仅2053处的替换就使病毒局部积累、系统传播速度和系统滴度提高了几倍。
