Department of Embryology, The Carnegie Institution of Washington, 115 W, University Pkwy, Baltimore, MD 21210, USA.
EMBO J. 1989 Apr;8(4):1023-32. doi: 10.1002/j.1460-2075.1989.tb03469.x.
The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The ;+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary ;-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.
单组分双生病毒（如玉米线条病毒，MSV）的整个基因组由约 2.7kb 的单链环状 DNA 组成。尽管这个大小仅足以编码三个平均大小的蛋白质，但该病毒能够引起许多单子叶植物的严重疾病，表现为黄化和矮化症状。我们已经确定了 MSV 感染过程中病毒系统传播和症状发展所必需的基因功能。通过定点突变和在侧翼 MSV 或包含在农杆菌 T-DNA 衍生载体中的报告基因序列之间的插入，创建了缺失和基因替换突变体。在农杆菌介导的玉米幼苗接种后，突变的 MSV DNA 通过侧翼序列内的同源重组从这些二元载体中被切除。我们的分析表明，MSV 的衣壳基因虽然不是复制所必需的，但对系统传播和随后的疾病发展是必不可少的。位于衣壳 ORF 上游的 ;+'链开放阅读框（ORF），预测编码 10.9kd 的蛋白质，也被发现对复制是可有可无的，但对系统传播是必不可少的。通过这种分析，支持自主复制的 MSV 序列被定位到包含两个病毒内含子区域和两个重叠互补 ;-'链 ORF 的 1.7kb 片段。尽管基因替换突变体不能系统传播，但接种和新发育的叶片都显示出与玉米某些发育突变体观察到的表型相似的黄化模式。讨论了 MSV 突变体表型与这些发育突变体的相似性。
