We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and beta-glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone.

病毒诱导的基因沉默（Virus-induced gene silencing，VIGS）是一种RNA 介导的植物抗病毒机制，近年来已被用作研究植物基因功能的反向遗传学手段。与转基因、基因敲除、反义抑制等基因功能研究方法相比，VIGS 技术研究周期短，不需要遗传转化，具有低成本、高通量等优势。因此，VIGS已被广泛应用于植物抗病抗逆、生长发育以及代谢调控等生理途径相关基因的功能鉴定。综述了VIGS 的机制、技术发展、沉默效率的影响因素及其在植物基因功能鉴定中的应用，并对VIGS 在植物性状改良及植物保护方面的应用前景进行了展望。

The phytoene desaturase (PDS) gene of Nicotiana benthamiana was silenced in plants infected with potato virus X (PVX) vectors carrying PDS inserts, and a green fluorescent protein (GFP) transgene was silenced in plants infected with PVX-GFP. This virus-induced gene silencing (VIGS) is post-transcriptional and cytoplasmic because it is targeted against exons rather than introns of PDS RNA and against viral RNAs. Although PDS and GFP RNAs are most likely targeted through the same mechanism, the VIGS phenotypes differed in two respects. PDS mRNA was targeted by VIGS in all green tissue of the PVX-PDS-infected plant, whereas PVX-PDS was not affected. In contrast, VIGS of the GFP was targeted against PVX-GFP. Initially, VIGS of the GFP was initiated in all green tissues, as occurred with PDS VIGS. However, after 30 days of infection, the GFP VIGS was no longer initiated in newly emerging leaves, although it was maintained in tissue in which it had already been initiated. Based on these analyses, we propose a model for VIGS in which the initiation of VIGS is dependent on the virus and maintenance of it is virus independent.

Research in plant science laboratories often involves usage of many different species, cultivars, ecotypes, mutants, alleles or transgenic lines. This creates a great challenge to keep track of the identity of experimental plants and stored samples or seeds.

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar 'Deltapine 5415' bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30 degrees C/26 degrees C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22 degrees C/18 degrees C. However, endogenous gene silencing decreased at 30 degrees C/26 degrees C. There was an approximately 7 d delay in the onset of gene silencing at 22 degrees C/18 degrees C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.

Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

SlDREB2 基因是利用rd29A 基因启动子的DRE 元件通过酵母单杂交技术从番茄幼苗（丽春）cDNA 文库中筛选得到的属于EREBP 家族的一个转录因子基因。利用构建病毒VIGS 载体，分别用含pBINTRB6、pTV00∷SlDREB2-1 和pTV00∷SlDREB2-2 重组载体的农杆菌GV3101 菌液灌根与叶片注射法侵染番茄植株，转化7 d 后进行病毒DNA 检测与验证。测定了干旱胁迫下侵染成功的番茄植株与野生型株系的生理指标，并通过Real-time qPCR 分析了VIGS 介导的SlDREB2 转录因子基因的表达特征。番茄植株干旱3 周胁迫处理后测定结果表明，野生番茄中脯氨酸和可溶性糖水平要高于被VIGS 病毒侵染的番茄植株，而丙二醛水平要低于被侵染植株，并且指标显示被pTV00∷SlDREB2-2 重组载体的农杆菌GV3101 菌液侵染的番茄植株抗旱性明显降低；复水1 周后，受VIGS 浸染株系丙二醛含量明显高于野生型，而可溶性糖与脯氨酸累积却低于野生型。Real-time qPCR 结果表明，经VIGS 病毒侵染过的番茄植株在胁迫处理时期SlDREB2 基因的相对表达量都明显低于野生型株系，表明番茄SlDREB2基因保守域末端459 bp 的特异片段具有较显著的沉默效果，说明VIGS 介导的SlDREB2 基因的表达受干旱诱导，该基因可以作为抗旱型作物品种改良的有价值基因。

对近年来病毒诱导的基因沉默（virus-induced gene silencing，VIGS）技术在番茄果实发育、激素调控、植物—病原物互作、防御害虫及非生物胁迫应答相关基因功能研究方面的进展进行了综述。

\n While RNA silencing is a potent antiviral defense in plants, well-adapted plant viruses are known to encode suppressors of RNA silencing (VSR) that can neutralize the effectiveness of RNA silencing. As a result, most plant genes involved in antiviral silencing were identified by using debilitated viruses lacking silencing suppression capabilities. Therefore, it remains to be resolved whether RNA silencing plays a significant part in defending plants against wild-type viruses. We report here that, at a higher plant growth temperature (26°C) that permits rigorous replication of\n Turnip crinkle virus\n (TCV) in\n Arabidopsis\n, plants containing loss-of-function mutations within the\n Dicer-like 2\n (\n DCL2\n ),\n Argonaute 2\n (\n AGO2\n ), and\n HEN1\n RNA methyltransferase genes died of TCV infection, whereas the wild-type Col-0 plants survived to produce viable seeds. To account for the critical role of DCL2 in ensuring the survival of wild-type plants, we established that higher temperature upregulates the activity of DCL2 to produce viral 22-nucleotide (nt) small interfering RNAs (vsRNAs). We further demonstrated that DCL2-produced 22-nt vsRNAs were fully capable of silencing target genes, but that this activity was suppressed by the TCV VSR. Finally, we provide additional evidence supporting the notion that TCV VSR suppresses RNA silencing through directly interacting with AGO2. Together, these results have revealed a specialized RNA silencing pathway involving DCL2, AGO2, and HEN1 that provides the host plants with a competitive edge against adapted viruses under environmental conditions that facilitates robust virus reproduction.\n

\n SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from\n Arabidopsis thaliana\n, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the\n Nicotiana benthamiana\n homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.\n

Antiviral defense of plants is usually enhanced by an elevated temperature under natural conditions. In order to better understand this phenomenon, we carried out temperature shift experiments with Nicotiana glutinosa plants that were infected with Potato virus X (PVX) or the necrotic strain of Potato virus Y (PVY(N)). The virus titer of the plants was found to be much lower when they were maintained at 30°C compared with 22°C, particularly in the upper leaves. PVX resistance at 30°C persisted for a short period even when temperature was shifted back to 22°C. In contrast, N. benthamiana lost the virus resistance immediately after the temperature dropped to 22°C. Expression analysis of two RNA-dependent RNA polymerases in N. glutinosa (NgRDR) showed that a 12-day treatment at 30°C increased the expression of NgRDR1, while NgRDR6 was not affected. In addition, the NgRDR6 mRNA level correlated with the PVX titer but was unaffected by PVY(N) infection. These observations indicate that PVX and PVY(N), although they are both RNA viruses, might trigger different defense responses at elevated temperatures. Our study provides valuable data for a better understanding of the temperature-regulated host virus interaction.

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar 'Deltapine 5415' bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30 degrees C/26 degrees C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22 degrees C/18 degrees C. However, endogenous gene silencing decreased at 30 degrees C/26 degrees C. There was an approximately 7 d delay in the onset of gene silencing at 22 degrees C/18 degrees C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.