Maize rayado fino virus (MRFV) is the type species of the genus Marafivirus in the family Tymoviridae. It infects maize (Zea mays), its natural host, to which it is transmitted by leafhoppers including Dalbulus maidis and Graminella nigrifrons in a persistent‐propagative manner. The MRFV monopartite RNA genome encodes a precursor polyprotein that is processed into replication‐associated proteins. The genome is encapsidated by two carboxy co‐terminal coat proteins, CP1 and CP2. Cloned MRFV can be readily transmitted to maize by vascular puncture inoculation (VPI), and such virus systems that can be used in maize are valuable to examine plant gene function by gene silencing. However, the efficacy of marafiviruses for virus‐induced gene silencing (VIGS) has not been investigated to date. To this end, MRFV genomic loci were tested for their potential to host foreign insertions without attenuating virus viability. This was done using infectious MRFV clones engineered to carry maize phytoene desaturase (PDS) gene fragments (ZmPDS) at various genomic regions. Several MRFV‐PDS constructs were generated and tested for infectivity and VIGS in maize. This culminated in identification of the helicase/polymerase (HEL/POL) junction as a viable insertion site that preserved virus infectivity, as well as several sites at which sequence insertion caused loss of virus infectivity. Transcripts of viable constructs, carrying PDS inserts in the HEL/POL junction, induced stable local and systemic MRFV symptoms similar to wild‐type infections, and triggered PDS VIGS initiating in veins and spreading into both inoculated and noninoculated leaves. These constructs were remarkably stable, retaining inserted sequences for at least four VPI passages while maintaining transmissibility by D. maidis. Our data thus identify the MRFV HEL/POL junction as an insertion site useful for gene silencing in maize.

中文翻译：

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工程玉米rayado fino病毒，用于病毒诱导的基因沉默。

玉米rayado菲诺病毒（MRFV）是属的模式种Marafivirus家庭芜菁发黄镶嵌病毒科。它感染玉米的天然宿主（玉米（Zea mays）），并由叶蝉（Dalbulus maidis）和黑粉虱（Graminella nigrifrons）传播给玉米以持续传播的方式。MRFV单部分RNA基因组编码前体多蛋白，该多蛋白被加工成复制相关蛋白。基因组被两个羧基共末端外壳蛋白CP1和CP2包裹。克隆的MRFV可以通过血管穿刺接种（VPI）轻易地传播给玉米，并且这种可用于玉米的病毒系统对于通过基因沉默检查植物基因功能非常有价值。但是，迄今为止尚未研究过马拉病毒对病毒诱导的基因沉默（VIGS）的功效。为此，测试了MRFV基因组基因座在不减弱病毒生存力的情况下携带外源插入物的潜力。这是通过使用可感染的MRFV克隆完成的，该克隆经过工程设计可在各种基因组区域携带玉米八氢番茄红素去饱和酶（PDS）基因片段（ZmPDS）。生成了几个MRFV-PDS构建体，并测试了玉米中的传染性和VIGS。最终鉴定出解旋酶/聚合酶（HEL / POL）连接是保留病毒感染性的可行插入位点，以及几个插入序列导致病毒感染性丧失的位点。可行构建体的转录本，在HEL / POL连接处带有PDS插入片段，可诱导类似于野生型感染的稳定的局部和全身MRFV症状，并触发PDS VIGS在静脉中起始并传播到接种和未接种的叶片中。这些构建体非常稳定，保留了至少四个VPI通道的插入序列，同时通过 最终鉴定出解旋酶/聚合酶（HEL / POL）连接是保留病毒感染性的可行插入位点，以及几个插入序列导致病毒感染性丧失的位点。可行构建体的转录本，在HEL / POL连接处带有PDS插入片段，可诱导类似于野生型感染的稳定的局部和全身MRFV症状，并触发PDS VIGS在静脉中起始并传播到接种和未接种的叶片中。这些构建体非常稳定，保留了至少四个VPI通道的插入序列，同时通过 最终鉴定出解旋酶/聚合酶（HEL / POL）连接是保留病毒感染性的可行插入位点，以及几个插入序列导致病毒感染性丧失的位点。可行构建体的转录本，在HEL / POL连接处带有PDS插入片段，可诱导类似于野生型感染的稳定的局部和全身MRFV症状，并触发PDS VIGS在静脉中起始并传播到接种和未接种的叶片中。这些构建体非常稳定，保留了至少四个VPI通道的插入序列，同时通过 诱导类似于野生型感染的稳定的局部和全身性MRFV症状，并触发PDS VIGS在静脉中起始并传播到接种和未接种的叶片中。这些构建体非常稳定，保留了至少四个VPI通道的插入序列，同时通过 诱导类似于野生型感染的稳定的局部和全身性MRFV症状，并触发PDS VIGS在静脉中启动，并传播到接种和未接种的叶片中。这些构建体非常稳定，保留了至少四个VPI通道的插入序列，同时通过D. maidis。因此，我们的数据将MRFV HEL / POL连接鉴定为可用于玉米基因沉默的插入位点。