Molecular analyzes of the mechanism of plant virus pathogenicity

我们对各种植物病毒的基因组复制机制进行了全面的研究。对花椰菜花叶病毒(CaMV)侵染的萝卜原生质体中病毒基因产物积累的时间序列分析表明，每个基因的表达受到不同的调控。亚基因组RNA和多顺反子35S RNA参与了CaMV基因的表达，表明CaMV在病毒增殖过程中采用了一种独特的基因表达策略。烟草花叶病毒(TMV)-OM的180K蛋白基因的两个结构域都含有甲基转移酶或RNA聚合酶的序列基序，具有与TMV-RNA结合的活性。利用针对这两个结构域的抗体进行免疫亲和层析，从烟草花叶病毒侵染的烟草叶片中分离到由130K和180K病毒蛋白组成的异源二聚体，它们在体外表现出依赖TMV-RNA的RNA合成。我们分析了拟南芥命名为um1和um2的突变体，其中黄瓜花叶病毒(CMV)的增殖被延迟。此外，我们一直在尝试通过定位克隆的方法来克隆CUM1基因。我们从接种马铃薯病毒V(PVX)的烟草原生质体中分离到了合成亚基因组RNA的可溶性RNA聚合酶。番茄花叶烟草花叶病毒(ToMV)130 kDa和180 kDa蛋白中的979位氨基酸在攻克番茄TM-1抗性基因中起重要作用。在ToMV蛋白中引入单一氨基酸替换(Gln979到Ile)，使突变体几乎不能在番茄细胞中复制，而在烟草细胞中完全复制，这表明氨基酸979周围的区域在宿主特异性决定因素方面的重要性。

We have comprehensively studied the mechanism of genome replication of various plant viruses. Chronological analyzes of the accumulation of viral gene products in cauliflower mosaic virus (CaMV)-infected turnip protoplasts showed that the expression of each gene is differentially regulated. It has also been suggested that subgenomic RNAs as well as the polycistronic 35S RNA are involved in the expression of CaMV genes, indicating that CaMV uses a unique strategy for the gene expression during virus multiplication. Two domains of the 180K protein gene of tobacco mosaic virus (TMV)-OM,each containing the sequence motifs of methyltransferase or RNA polymerase, have shown to exhibit the binding activity with TMV-RNA.By immunoaffinity chromatography using antibodies specfic to the domains, heterodimers consisting of 130K and 180K viral proteins, which exhibited TMV-RNA dependent RNA synthesis in vitro, were isolated from TMV-infected tobacco leaves. We have analyzed Arabidopsis thaliana mutants designated cum1 and cum2, in which the multiplication of cucumber mosaic virsu (CMV) is delayd. Furthermore, we have been trying to clone the CUM1 gene by the positional cloning procedure. We have isolated solubilized RNA polymerase synthesizing subgenomic RNA from tobacco protoplasts inoculated with potato virus V (PVX). Amino acid 979 of the 130-and 180-kDa proteins of tomato mosaic tobamovirus (ToMV) plays an important role in overcoming the tomato Tm-1 resistance gene. Introduction of a single amino acid substitution (Gln979 to Ile) into the ToMV proteins rendered the mutant virtually unable to replicate in tomato cells while it replicated perfectly in tobacco cells, indicating the importance of the region surrounding the amino acid 979 in terms of host specificity determinants.