马铃薯Y病毒P3N-PIPO与马铃薯BI-1互作的生物学功能解析

In recent years, PIPO, which is identified as a newer ORF of the potato virus Y, has been widely concerned. The previous study found that PIPO was a virus motility-related protein, then it was reported that PIPO was involved in resistance-breaking and symptom expression. Although the interactions between PIPO and the host proteins had been studied in many different virus-host pathogenic systems, the PIPO functions were still unclear and conflicting, which indicated that PIPO, like other viral proteins, has multiple functions during the viral infection. There were 7 proteins of potato interacted with PVY P3NPIPO in our previous research, One of them, encoded by BI-1 gene, is a widely-conserved cell apoptosis inhibiting factor in plant, animal and yeast. In this project, we want to study: (1)was the P3N-PIPO involved in the regulation of host cell apoptosis process? (2)was BI-1 involved in the PVY transportation between cell cytoplasm and plasmodesma? (3)What is the effect of the host BI-1 gene modification on PVY movement and replication? In a word, we try to carry out some researches on the co-location of interacting proteins, cell apoptosis inhibition, virus movement inhibition, and the genetic modification of host BI-1, to reveal the biological role of interaction between the P3N-PIPO and BI-1.

关于马铃薯Y病毒属病毒基因组中一个最近被鉴定的ORF PIPO近年来被广泛的关注。最初的研究发现PIPO是一个运动相关蛋白，随后有报道PIPO参与了抗性突破和症状表达。目前有关PIPO与宿主蛋白的互作研究在不同的病毒/寄主系统中所得结果有所不同，说明PIPO和其他病毒蛋白一样，在病毒致病过程中具有多种功能。前期研究工作中，我们筛选到7个与PVY P3NPIPO互作的马铃薯蛋白。其中BI-1基因编码一个在动植物和酵母中具有广泛保守性的细胞凋亡抑制因子。本项目拟将开展互作蛋白共定位、细胞凋亡抑制试验、病毒运动抑制试验、宿主BI-1基因修饰等研究内容来解析P3N-PIPO与马铃薯BI-1互作产生的生物学意义，拟解决的科学问题包括：（1）P3N-PIPO是否参与调控宿主细胞凋亡的进程？（2）BI-1是否参与PVY在马铃薯胞质或胞间连丝中的运输？（3）对宿主BI-1基因的修饰对病毒运动或复制的影响。

自噬参与植物的生长发育，也是植物对抗病毒侵染的一种防卫机制，病毒也能劫持自噬途径以利于自身的复制。. 我们前期的研究工作证实PVY P3N-PIPO蛋白与马铃薯及本氏烟的BI-1蛋白有很强的互作关系，BI-1基因是一个在动植物及微生物中广泛存在的保守的细胞凋亡抑制因子。. 本研究中利用CRISPR-Cas9基因编辑技术和RNA干扰技术分别获得了本氏烟的NbBI-1缺失突变株和干扰突变株。. PVYN-Wi侵染本氏烟野生型14天时，叶片细胞自噬活性明显增强， 与健康的野生型本氏烟相比，叶片中NbATG6（一种自噬核心蛋白）RNA的表达量下降，叶片病毒累积量升高明显，叶片皱缩卷曲明显；而PVY侵染BI-1缺失突变体后，NbATG6 RNA表达量与健康的BI-1缺失突变体没有明显差异，叶片细胞自噬活性与PVY感染野生型本氏烟相比降低，病毒积累量下降，叶片皱缩卷曲症状轻微。. BI-1敲除的本氏烟，生长比野生型快，高叶绿素含量增加，光合作用增强，但耐旱性和耐寒性都下降。. 在本氏烟叶片上分别瞬时表达P3、P3N-PIPO、GFP（阴性对照）和Bax（一种小鼠自噬蛋白，作为阳性对照）蛋白，P3和P3N-PIPO能引起与Bax类似的自噬反应；上述4种蛋白在BI-1缺失突变体叶片上瞬时表达，P3、P3N-PIPO、GFP和Bax均未引起自噬反应。. PVY NIb被单独瞬时表达或与NbATG6共同瞬时表达在野生型本氏烟叶片上，发现NIb的含量相对NIb单独表达时有所下降。. 据此我们推测，PVYN-Wi侵染本氏烟时，其编码的P3和P3N-PIPO会通过与NbBI-1互作，抑制NbATG6的表达，降低NbATG6浓度，从而减少对PVY NIb的降解，促进了病毒复制和增值。