【作者】 卓涛；

【导师】 韩成贵；

【作者基本信息】 中国农业大学 ， 植物病理学， 2014， 博士

【摘要】 马铃薯卷叶病毒(PLRV)分布广泛,主要在马铃薯植株上造成危害,导致严重的经济损失。PLRV是黄症病毒科,马铃薯卷叶病毒属的代表种。此病毒属的病毒含有6个ORF,编码6种蛋白,其中的P0蛋白以抑制子的功能为大家所知。近年来,虽然有大量的此属病毒P0抑制子的研究报道,而关于此蛋白抑制基因沉默的作用机制仍然不十分清楚。本研究的对象是PLRV内蒙古分离物P0(以下简称POPL-IM),通过农杆菌共浸润瞬时表达系统确认了其是一个强的RNA沉默抑制子。通过POPI-IM与之前报道的弱抑制子PLRV荷兰分离物PO (POPI-NL)氨基酸序列比对,发现两种PO之间仅有五个氨基酸的差异,在对POPL-IM的这五个氨基酸互换突变体鉴定发现,这些单突变体对抑制子的活性未产生影响。同时,将这五个氨基酸同时进行了互换突变,此突变体氨基酸序列与POPI-NL完全一致,其抑制子的活性与野生型的POPL-IM没有差异。将这五个氨基酸分别突变成A,发现第102位的S和第228位的Ⅰ突变成A后,导致抑制子活性降低。本研究第一次明确了PLRV PO的类似F-box的模体为第76-95位的76LPRHLHYECLEWGLLCGTHP95,而不是之前报道的第59-77位的区域,并且证明了第76位的L一旦突变成A后,其抑制子活性完全丧失。在对马铃薯卷叶病毒属PO的C端FWR保守序列的验证中,发现POPL-IM第220位F氨基酸突变成R后,由于氨基酸极性改变也使得PO的抑制子活性丧失。本研究还对POPL-IM中马铃薯卷叶病毒属其它抑制子PO所没有的类似WG/GW的模体--W87/G88和G139/W140/G141进行了功能鉴定。其中,前一个WG序列位于类似F-box的模体中,其任何一个氨基酸的突变或者互换都使POPL-IM不能发挥抑制子功能。而后一个GWG序列,只有将其中的W突变成A后,POPI-LM抑制子活性几乎完全丧失,其它的A替换突变体或者三个氨基酸相互换位,POPL-IM抑制子活性则都不受影响。对PoPL-IN及其突变体进行生物学检测,发现POPL-IM抑制了次级siRNA的产生。对POPL-IM作用机制进行初步的研究,发现POPL-IM和具有抑制活性的突变体能够介导寄主蛋白AGO1的降解。由于有较多关于马铃薯卷叶病毒P0通过类似F-box的模体与SKP1互作的报道,但是本研究的实验结果显示,无论在酵母中还是在植物体中,POPL-IM都不与NbSKPl发生互作。同时,本文还鉴定了豌豆轻型褪绿病毒(PMCV)中国分离物编码的PO蛋白具有强的RNA沉默抑制功能,并且确定了影响其抑制子活性的关键氨基酸第62、63位LP基序。此结果再次证明了马铃薯卷叶病毒属PO中类似F-box的模体的重要性。PMCV的PO是已知的马铃薯卷叶病毒属中最大的P0蛋白,其全长为271aa,因此对其N端和C端分别进行缺失突变,发现其N端缺失20aa完全破坏了此蛋白抑制子的活性,而C末端缺失41aa仍然保持了抑制子活性,说明了N端氨基酸对其抑制子功能的重要性。本论文主要是对马铃薯卷叶病毒属两种病毒的P0蛋白进行了抑制子功能分析,并且定位了影响抑制子功能的关键氨基酸,特别是PLRV PO中还发现了此属病毒的抑制子P0没有的类似WG/GW的模体。这些定位结果以及对POPI-IM作用机制的初步研究结果,丰富了马铃薯卷叶病毒属P0抑制子功能的多样性,同时也为P0作用机理的最终揭示提供了重要参考数据。更多还原

【Abstract】 Potato leafroll virus (PLRV) is distributed worldwide and infects economically important potato crop, causing important loss in agriculture. PLRV which belongs to the Luteoviridae family, is the type member of the Polerovirus genus and the genome consists of single-stranded plus-sense RNA with six recognized ORFs (open reading frames). The PO proteins encoded by the5’-proximal ORF of poleroviruses are well known RNA silencing suppressors. Recently, many PO-related studies have been reported, but the mechanism of PO remains unclear. In our study, we used Agrobacterium infiltration-mediated RNA silencing assays to establish that the Inner Mongolian PLRV PO protein (POPL-IM) has a strong suppressor activity. Five amino-acid substitutions in POPL-IM with those corresponding to the PO protein of a Netherlandish isolate of PLRV (POPL-NL) did not affect the strong suppressor activity, implying that the distinction between the two PO proteins was independent of the differences between their amino acids. But the mutations to A among the different amino acids, S102A and I228A, affect its suppressor activity. Strikingly, poPL-IM has an unusual F-box-like motif that contains a Trp/Gly sequence (87W/88G) and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other PO proteins, and contains two POPL-IM LP motif signature residues, L59/P60and L76/P77, matched an F-box-like domain. Mutagenesis experiments demonstrated that the POPL-IM F-box-like motif encompasses amino acids76-LPRHLHYECLEWGLLCGTHP-95, and that the suppressor activity is abolished by L76A, W87A or G88A substitution. The suppressor activity is also weakened substantially by mutations within the G139/W140/G141region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of POPL-IM. We conducted the alignment of POPL from various isolates and found the motifs (76LPRHLHYECLEWGLLCGTHP95, G139WG140and F220) were very conserved and had no variation and the results of mutagenesis experiments also comfirmed these motifs are very important to POPL suppressor activities. As has been observed with other PO proteins, the suppression of POPL-IM is correlated with the reducion of the accumulation of host AGO1silencing complex protein. However, POPL-IM fails to bind Nicotiana benthamiana SKP1, which functions in a proteasome pathway that may be involved in AGO1degradation, either in yeast or in plants. These results suggest that P0PL-IM may suppress RNA silencing by using an alternative pathway to target AGO1for degradation.In our studies, we also identified another strong RNA silencing suppressor, the PO protein of Pea mild chlorisis virus (PMCV) and also found a F-box-like motif in PMCV PO. The LP motif signature residues, L62/P63, contributed to the suppressor activity. The PMCV PO is the longest known PO protein among polerovirues, which has271amino acids. Deletion of the first20N-terminal amino acid residues of PMCV PO destroyed suppression of RNA silencing, but the41C-terminal amino acid residue region of PMCV PO was not required for silencing suppressor activity.All of the results enrich the diversity of POs of poleroviruses as suppressors and help improve our understanding of the molecular mechanisms involved in poleroviruses infection.更多还原