Transient expression of HPV16 E7 peptide (aa 44-60) and HPV16 L2 peptide (aa 108-120) on chimeric potyvirus-like particles using Potato virus X-based vector

The optimized expression of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV16) was developed. Epitope derived from minor capsid protein L2 was expressed as N-terminal fusion with ACP while an epitope derived from E7 oncoprotein was fused to its C-terminus. The construct was cloned into Potato X potexvirus (PVX) based vector and transiently expressed in plants using Agrobacterium tumefaciens mediated inoculation. To increase the level of expressed protein the transgenic Nicotiana benthamiana plants expressing Potato virus A HC-Pro gene and transgenic Nicotiana tabacum, cv. Petit Havana SR1 carrying Potato virus A P3 protein gene were tested. Synergistic infection of host plants with PVX carrying the construct and Potato virus Y(O) (PVY(O)) increased the expression of L2ACPE7 in N. tabacum and in transgenic N. benthamiana carrying potyviral HC-Pro gene as compared to control plants infected with L2ACPE7 only.     

A high-efficiency gene silencing in plants using two-hit asymmetrical artificial MicroRNAs

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. They are produced through an enzyme-guided process called dicing and have an asymmetrical structure with two nucleotide overhangs at the 3′ ends. Artificial microRNAs (amiRNAs or amiRs) are designed to mimic the structure of miRNAs and can be used to silence specific genes of interest. Traditionally, amiRNAs are designed based on an endogenous miRNA precursor with certain mismatches at specific positions to increase their efficiency. In this study, the authors modified the highly expressed miR168a in Arabidopsis thaliana by replacing the single miR168 stem-loop/duplex with tandem asymmetrical amiRNA duplexes that follow the statistical rules of miRNA secondary structures. These tandem amiRNA duplexes, called “two-hit” amiRNAs, were shown to have a higher efficiency in silencing GFP and endogenous PDS reporter genes compared to traditional “one-hit” amiRNAs. The authors also demonstrated the effectiveness of “two-hit” amiRNAs in silencing genes involved in miRNA, tasiRNA, and hormone signalling pathways, individually or in families. Importantly, “two-hit” amiRNAs were also able to over-express endogenous miRNAs for their functions. The authors compare “two-hit” amiRNA technology with CRISPR/Cas9 and provide a web-based amiRNA designer for easy design and wide application in plants and even animals.     

Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69(159) and amiR-HC-Pro(159) are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP(159) targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69(159) and amiR-HC-Pro(159) from a dimeric pre-amiR-P69(159)/amiR-HC-Pro(159) transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 degrees C, a temperature that compromises small interfering RNA-mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.     

MicroRNAs with analogous target complementarities perform with highly variable efficacies in Arabidopsis

In plants, the silencing efficacy of microRNAs (miRNAs) is thought to be predominantly determined by the degree of complementarity to their target genes. Here, silencing efficacy was determined for Arabidopsis miR159 and four artificial miRNAs (amiRNAs) that all target MYB33/MYB65 with analogous complementarities. As determined through complementation of a loss-of-function mir159 mutant, the amiRNAs displayed highly variable efficacies, none of which was as strong as endogenous miR159. This was despite amiRNA expression levels being many fold-higher than miR159 in wild-type. The results highlight the variable nature of miRNA silencing efficacy in plants, where it appears that factors additional to complementarity strongly impact silencing.     

Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus

MicroRNAs (miRNAs) regulate the abundance of target mRNAs by guiding cleavage at sequence complementary regions. In this study, artificial miRNAs (amiRNAs) targeting conserved motifs of the L (replicase) gene of Watermelon silver mottle virus (WSMoV) were constructed using Arabidopsis pre-miRNA159a as the backbone. The constructs included six single amiRNAs targeting motifs A, B1, B2, C, D of E, and two triple amiRNAs targeting motifs AB1E or B2DC. Processing of pre-amiRNAs was confirmed by agro-infiltration, and transgenic Nicotiana benthamiana plants expressing each amiRNA were generated. Single amiRNA transgenic lines expressing amiR-LB2 or amiR-LD showed resistance to WSMoV by delaying symptom development. Triple amiRNA lines expressing amiR-LB2, amiR-LD and amiR-LC provided complete resistance against WSMoV, with no indication of infection 28 days after inoculation. Resistance levels were positively correlated with amiRNA expression levels in these single and triple amiRNA lines. The triple amiR-LAB1E line did not provide resistance to WSMoV. Similarly, the poorly expressed amiR-LC and amiR-LE lines did not provide resistance to WSMoV. The amiR-LA- and amiR-LB1-expressing lines were susceptible to WSMoV, and their additional susceptibility to the heterologous Turnip mosaic virus harbouring individual target sequences indicated that these two amiRNAs have no effect in vivo. Transgenic lines expressing amiR-LB2 exhibited delayed symptoms after challenge with Peanut bud necrosis virus having a single mismatch in the target site. Overall, our results indicate that two amiRNAs, amiR-LB2 and amiR-LD, of the six designed amiRNAs confer moderate resistance against WSMoV, and the triple construct including the two amiRNAs provides complete resistance.     

马铃薯Y病毒蚜传辅助因子促进马铃薯X病毒长距离运输

采用PCR和定点突变法,对马铃薯Y病毒中国株系（Chyinese strain of potato Ypotyvirus,PVY-C)蚜传辅助成分（helper component proteinase,HC-Pro)基因中心区域的CCCT基序和PTK基序进行定点改造,获得了4种突变体。然后将突变体砍降到植物表达载体pBin438中,所得到的重组体通过根癌土壤杆菌（Agrobacterium tumefaciens(Smith et Townsend)Conn）介导法转了烟草（Nicotiana tabacum L.cv.K326).Southern blotting和Western blotting分析表明4种突变体已经成功整合到烟草的基因组中,并在蛋白水平上得到了表达。马铃薯X病毒（potato X potexvirus,PVX)对转基因烟草的攻毒实验表明,4种突变体均使PVY－C HYC－Prog严重丧失了促进PVX病毒粒子在寄主体内积累和提高PVX致病性的功能,说明CCCT、PTK基序为PVY－C HYC－Pro介导PVX／PVY协生作用所必需。同时证明了HC-Pro具有增强PVX在寄主体内长距离运输的功能。     

Heterologous expression of artificial miRNAs from rice dwarf virus in transgenic rice

In contrast to hairpin RNAs, in which heterogeneous small RNAs are processed from double-stranded RNA to have potential off-target effects on endogenous other genes, artificial miRNAs (amiRNAs) have advantages of exquisite specificity and non-transitivity to thus target individual genes and groups of endogenous genes. Earlier studies showed that amiRNA engineering based on osa-miRNA528 precursor could efficiently trigger endogenous gene silencing and modulate agronomic traits in rice. However, both the expression efficiency of heterologous amiRNAs based on osa-miRNA528 precursor and the correlation of copy number with the relative expression level of amiRNAs remain unknown. In the present study, five amiRNAs (S9-1174, S9-1192, S11-864, S11-868 and S11-869) targeting different sites of S9 and S11 negative strands in rice dwarf virus (RDV) genome were constructed using endogenous osa-miRNA528 precursor as backbone. After identification by Northern blot, two amiRNAs (S9-1174 and S9-1192) targeting S9 negative strand in RDV genome were highly expressed, whereas in three tested amiRNAs targeting S11 negative strand in RDV genome, only two amiRNAs (S11-868 and S11-869) were processed efficiently. T0 generation transgenic rice containing amiRNAs (S9-1174, S9-1192, S11-868 and S11-869) exhibited different expression ratios of amiRNAs, accounting for 90.0, 90.0, 66.7 and 77.8 %, respectively. In addition, combination analysis with the relative amiRNA expression levels and its copy number revealed that the relative expression levels of amiRNAs had no relation to the copy number of T-DNA insert in transgenic rice.     

Potato Y Potyvirus Helper Component Proteinase Enhances Long-distance Movement of Potato X Potexvirus

To mutagenize two conserved CCCT and PTK motifs in the central domain of Chinese strain of potato Y potyvirus (PVY-C) helper component proteinase (HC-Pro), four mutants of HC-Pro gene were obtained by PCR and site-directed mutagenesis, and then were inserted into the constitutive expression vector pBin438. Leaves from tobacco ( Nicotiana tabacum L. cv. K326) were transformed with these four plant expression plasmids by Agrobacterium -mediated transformation, respectively. Southern and Western blotting analyses showed that these four mutants were integrated into tobacco genomic DNA and could express the corresponding proteins in most of the transgenic plants. The challenge of transgenic plants with potato X potexvirus (PVX) revealed that the expression products of PVY-C HC-Pro mutants in transgenic plants greatly abolished functions of HC-Pro in enhancing the accumulation and pathogenicity of PVX, indicating that CCCT and PTK motifs of HC-Pro were required for PVX/PVY synergism. Meanwhile, the results demonstrated that PVY-C HC-Pro had a function in accelerating the long-distance movement of PVX in these transgenic plants for the first time.     

An in vivo Transient Expression System Can Be Applied for Rapid and Effective Selection of Artificial MicroRNA Constructs for Plant Stable Genetic Transformation

The utility of artificial microRNAs (amiRNAs) to induce loss of gene function has been reported for many plant species, but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable. In this study, expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation. A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments. Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs. This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants.     

基于烟草瞬时表达体系对amiRNA沉默效果快速有效的预验证

amiRNA(artificial microRNA)作为一种诱导基因发生特异性沉默的技术已在多种植物中应用,但设计出的不同amiRNAs在所转化株系中的沉默效率难以预测,因此对amiRNA载体的沉默效率进行预验证是非常必要的。本实验以丹参(Salvia miltiorrhiza)的1个MYB类转录因子基因SmPAP1的mRNA序列为amiRNA作用对象,并挑选2个经在线软件WMD3(Web MicroRNA Designer)设计的amiRNAs,分别命名为amiRNA1-SmPAP1和amiRNA2-SmPAP1,然后通过农杆菌介导将构建的2个amiRNA载体和SmPAP1过表达植物载体在烟草叶片细胞中进行瞬时共表达。结果显示,amiRNA2的表达丰度约是amiRNA1的2倍;amiRNA2对靶标SmPAP1的沉默效率约是amiRNA1的2.5倍;SmPAP1在mRNA和蛋白水平上均与相应amiRNA的表达水平呈显著负相关。因此,amiRNA在烟草细胞中的瞬时表达可快速、有效地对不同amiRNA沉默效果进行预验证,从而为后续的植物遗传转化研究提供重要参考。     

Resistance to Wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat

Wheat streak mosaic virus (WSMV) is a persistent threat to wheat production, necessitating novel approaches for protection. We developed an artificial miRNA strategy against WSMV, incorporating five amiRNAs within one polycistronic amiRNA precursor. Using miRNA sequence and folding rules, we chose five amiRNAs targeting conserved regions of WSMV but avoiding off-targets in wheat. These replaced the natural miRNA in each of five arms of the polycistronic rice miR395, producing amiRNA precursor, FanGuard (FGmiR395), which was transformed into wheat behind a constitutive promoter. Splinted ligation detected all five amiRNAs being processed in transgenic leaves. Resistance was assessed over two generations. Three types of response were observed in T(1) plants of different transgenic families: completely immune; initially resistant with resistance breaking down over time; and initially susceptible followed by plant recovery. Deep sequencing of small RNAs from inoculated leaves allowed the virus sequence to be assembled from an immune transgenic, susceptible transgenic, and susceptible non-transgenic plant; the amiRNA targets were fully conserved in all three isolates, indicating virus replication on some transgenics was not a result of mutational escape by the virus. For resistant families, the resistance segregated with the transgene. Analysis in the T(2) generation confirmed the inheritance of immunity and gave further insights into the other phenotypes. Stable resistant lines developed no symptoms and no virus by ELISA; this resistance was classified as immunity when extracts failed to transmit from inoculated leaves to test plants. This study demonstrates the utility of a polycistronic amiRNA strategy in wheat against WSMV.     

A simplified method for constructing artificial microRNAs based on the osa-MIR528 precursor

Artificial miRNAs (amiRNAs) have been used successfully in various plants to silence endogenous genes or viral RNAs. The method of Schwab et al., widely used to construct amiRNAs, requires four PCRs. We describe a simplified method of constructing amiRNA based on the osa-MIR528 backbone using one PCR step by addition of prolonging sequence to the primers. The length of prolonging sequence needed in the osa-MIR528 precursor was determined. Using this method, we constructed amiRNA targeting the Nicotiana benthamiana UPF1 gene and showed that it functioned in silencing UPF1 expression in leaves when expressed transiently.     

Characterization of the recombinant forms arising from a Potato virus X chimeric virus infection under RNA silencing pressure

Recombination is a frequent phenomenon in RNA viruses whose net result is largely influenced by selective pressures. RNA silencing in plants acts as a defense mechanism against viruses and can be used to engineer virus resistance. Here, we have investigated the influence of RNA silencing as a selective pressure to favor recombinants of PVX-HCT, a chimeric Potato virus X (PVX) vector carrying the helper-component proteinase (HC-Pro) gene from Plum pox virus (PPV). All the plants from two lines expressing a silenced HC-Pro transgene were completely resistant to PPV. However a significant proportion became infected with PVX-HCT. Analysis of viral RNAs accumulating in silenced plants revealed that PVX-HCT escaped silencing-based resistance by removal of the HC-Pro sequences that represented preferential targets for transgene-promoted silencing. The virus vector also tended to lose the HC-Pro insert when infecting transgenic plants containing a nonsilenced HC-Pro transgene or wild-type (wt) Nicotiana benthamiana plants. Nevertheless, loss of HC-Pro sequences was faster in nonsilenced transgenic plants than in wt plants, suggesting the transgene plays a role in promoting a higher selective pressure in favor of recombinant virus versions. These results indicate that the outcome of recombination processes depends on the strength of selection pressures applied to the virus.