Jing Qu

Artificial MicroRNA-Mediated Virus Resistance in Plants

ABSTRACT RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the hosts RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.

Development of marker-free transgenic Jatropha plants with increased levels of seed oleic acid

BackgroundJatropha curcas is recognized as a new energy crop due to the presence of the high amount of oil in its seeds that can be converted into biodiesel. The quality and performance of the biodiesel depends on the chemical composition of the fatty acids present in the oil. The fatty acids profile of the oil has a direct impact on ignition quality, heat of combustion and oxidative stability. An ideal biodiesel composition should have more monounsaturated fatty acids and less polyunsaturated acids. Jatropha seed oil contains 30% to 50% polyunsaturated fatty acids (mainly linoleic acid) which negatively impacts the oxidative stability and causes high rate of nitrogen oxides emission.ResultsThe enzyme 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in plants. We identified three putative delta12fatty acid desaturase genes in Jatropha (JcFAD2s) through genome-wide analysis and downregulated the expression of one of these genes, JcFAD2-1, in a seed-specific manner by RNA interference technology. The resulting JcFAD2-1 RNA interference transgenic plants showed a dramatic increase of oleic acid (> 78%) and a corresponding reduction in polyunsaturated fatty acids (< 3%) in its seed oil. The control Jatropha had around 37% oleic acid and 41% polyunsaturated fatty acids. This indicates that FAD2-1 is the major enzyme responsible for converting oleic acid to linoleic acid in Jatropha. Due to the changes in the fatty acids profile, the oil of the JcFAD2-1 RNA interference seed was estimated to yield a cetane number as high as 60.2, which is similar to the required cetane number for conventional premium diesel fuels (60) in Europe. The presence of high seed oleic acid did not have a negative impact on other Jatropha agronomic traits based on our preliminary data of the original plants under greenhouse conditions. Further, we developed a marker-free system to generate the transgenic Jatropha that will help reduce public concerns for environmental issues surrounding genetically modified plants.ConclusionIn this study we produced seed-specific JcFAD2-1 RNA interference transgenic Jatropha without a selectable marker. We successfully increased the proportion of oleic acid versus linoleic in Jatropha through genetic engineering, enhancing the quality of its oil.

Rapid analysis of Jatropha curcas gene functions by virus‐induced gene silencing

Jatropha curcas L. is a small, woody tree of the Euphorbiaceae family. This plant can grow on marginal land in the tropical and subtropical regions and produces seeds containing up to 30% oil. Several Asian countries have selected Jatropha for large scale planting as a biodiesel feedstock. Nevertheless, Jatropha also possesses several undesirable traits that may limit its wide adoption. An improved understanding of plant development and the regulation of fatty acid (FA) and triacylglyceride biosynthesis in Jatropha is particularly facilitative for the development of elite crops. Here, we show that a tobacco rattle virus (TRV) vector can trigger virus-induced gene silencing (VIGS) in Jatropha. Our optimized method produced robust and reliable gene silencing in plants agroinoculated with recombinant TRV harbouring Jatropha gene sequences. We used VIGS to investigate possible functions of 13 Jatropha genes of several functional categories, including FA biosynthesis, developmental regulation and toxin biosynthesis, etc. Based on the effects of VIGS on the FA composition of newly emerged leaves, we determined the function of several genes implicated in FA biosynthesis. Moreover, VIGS was able to discriminate independent functions of related gene family members. Our results show that VIGS can be used for high-throughput screening of Jatropha genes whose functions can be assayed in leaves.

Virulence Factors of Geminivirus Interact with MYC2 to Subvert Plant Resistance and Promote Vector Performance

To attract more disease vectors for viral transmission, geminivirus targets the plant transcription factor MYC2 to suppress plant terpene-based resistance against whitefly. A pathogen may cause infected plants to promote the performance of its transmitting vector, which accelerates the spread of the pathogen. This positive effect of a pathogen on its vector via their shared host plant is termed indirect mutualism. For example, terpene biosynthesis is suppressed in begomovirus-infected plants, leading to reduced plant resistance and enhanced performance of the whiteflies (Bemisia tabaci) that transmit these viruses. Although begomovirus-whitefly mutualism has been known, the underlying mechanism is still elusive. Here, we identified βC1 of Tomato yellow leaf curl China virus, a monopartite begomovirus, as the viral genetic factor that suppresses plant terpene biosynthesis. βC1 directly interacts with the basic helix-loop-helix transcription factor MYC2 to compromise the activation of MYC2-regulated terpene synthase genes, thereby reducing whitefly resistance. MYC2 associates with the bipartite begomoviral protein BV1, suggesting that MYC2 is an evolutionarily conserved target of begomoviruses for the suppression of terpene-based resistance and the promotion of vector performance. Our findings describe how this viral pathogen regulates host plant metabolism to establish mutualism with its insect vector.

A critical domain of the Cucumber mosaic virus 2b protein for RNA silencing suppressor activity

Alignment of Cucumber mosaic virus (CMV) 2b protein sequences from two CMV subgroups revealed two highly variable regions. To examine contributions of variable sequence domains to the suppressor activity, we performed a comparative study between 2b proteins of a subgroup I strain (SD‐CMV) and a subgroup II strain (Q‐CMV). Here we show that the suppressor activity of SD2b is stronger than that of Q2b and that a domain existent in SD2b but absent in Q2b is a major determinant of the suppressor activity of SD2b. We further show that the same domain is responsible for inhibition of Nicotiana benthamiana AGO4‐1 transcription. Our results implicate AGO4 as a mediator for CMV 2b to suppress systemic silencing and DNA methylation.

Dissecting functions of KATANIN and WRINKLED1 in cotton fiber development by virus-induced gene silencing.

Most of the world’s natural fiber comes from cotton (Gossypium spp.), which is an important crop worldwide. Characterizing genes that regulate cotton yield and fiber quality is expected to benefit the sustainable production of natural fiber. Although a huge number of expressed sequence tag sequences are now available in the public database, large-scale gene function analysis has been hampered by the low-efficiency process of generating transgenic cotton plants. Tobacco rattle virus (TRV) has recently been reported to trigger virus-induced gene silencing (VIGS) in cotton leaves. Here, we extended the utility of this method by showing that TRV-VIGS can operate in reproductive organs as well. We used this method to investigate the function of KATANIN and WRINKLED1 in cotton plant development. Cotton plants with suppressed KATANIN expression produced shorter fibers and elevated weight ratio of seed oil to endosperm. By contrast, silencing of WRINKLED1 expression resulted in increased fiber length but reduced oil seed content, suggesting the possibility to increase fiber length by repartitioning carbon flow. Our results provide evidence that the TRV-VIGS system can be used for rapid functional analysis of genes involved in cotton fiber development.

A new strain of Indian cassava mosaic virus causes a mosaic disease in the biodiesel crop Jatropha curcas

Jatropha curcas mosaic disease is a newly emerging disease that challenges the productivity of a prospective biofuel crop, J. curcas. The aetiology of this disease has not been resolved. Here, we report the complete nucleotide sequences of a Jatropha virus isolated from Dharwad, Southern India. Phylogenetic analysis of the virus genome suggests it is a new strain of Indian cassava mosaic virus. Agroinfiltration of the two cloned viral DNA components produced systemic infection and typical mosaic symptoms in J. curcas, thereby fulfilling Koch’s postulates. The availability of infectious clones will provide a valuable tool to screen J. curcas cultivars for disease resistance and facilitate the generation of virus-resistant J. curcas plants by transgenic technology.

The Jatropha FT ortholog is a systemic signal regulating growth and flowering time

BackgroundJatropha curcas is being promoted as a new bioenergy crop in tropical and subtropical regions due to its high amount of seed oil and its potential capacity to grow on marginal land for biofuel production. However, the productivity of the plant is constrained by the unfavorable flowering time and inflorescence architecture, which render harvesting of seeds time-consuming and labor-intensive. These flowering-related traits have limited further widespread cultivation of Jatropha.ResultsWe identified a Jatropha curcas homolog of Flowering locus T (JcFT) and demonstrated its function by genetic complementation of the Arabidopsis ft mutant. The JcFT expression level was found to be remarkably correlated with leaf age. Overexpression of JcFT in Jatropha reduced flowering time and altered plant architecture by producing more branches. Grafting experiments suggested that the earlyflowering and alteration of plant architecture traits were graft-transmissible. We also showed that the FT-overexpressing transgenic Jatropha can be used as a root stock for grafting of scions derived from other Jatropha.ConclusionWe generated early flowering transgenic Jatropha plants that accumulate higher levels of the florigen FT. Not only early flowering but also plant growth was affected in JcFT overexpression lines. More seeds can be produced in a shorter time frame by shortening the flowering time in Jatropha, suggesting the possibility to increase seed yield by manipulating the flowering time.

Engineering geminivirus resistance in Jatropha curcus

BackgroundJatropha curcus is a good candidate plant for biodiesel production in tropical and subtropical regions. However, J. curcus is susceptible to the geminivirus Indian cassava mosaic virus (ICMV), and frequent viral disease outbreaks severely limit productivity. Therefore the development of J. curcus to carry on durable virus resistance remains crucial and poses a major biotechnological challenge.ResultsWe generated transgenic J. curcus plants expressing a hairpin, double-stranded (ds) RNA with sequences homologous to five key genes of ICMV-Dha strain DNA-A, which silences sequence-related viral genes thereby conferring ICMV resistance. Two rounds of virus inoculation were conducted via vacuum infiltration of ICMV-Dha. The durability and heritability of resistance conferred by the dsRNA was further tested to ascertain that T1 progeny transgenic plants were resistant to the ICMV-SG strain, which shared 94.5% nucleotides identity with the ICMV-Dha strain. Quantitative PCR analysis showed that resistant transgenic lines had no detectable virus.ConclusionsIn this study we developed transgenic J. curcus plants to include a resistance to prevailing geminiviruses in Asia. These virus-resistant transgenic J. curcus plants can be used in various Jatropha breeding programs.

Geminivirus Activates ASYMMETRIC LEAVES 2 to Accelerate Cytoplasmic DCP2-Mediated mRNA Turnover and Weakens RNA Silencing in Arabidopsis.

Aberrant viral RNAs produced in infected plant cells serve as templates for the synthesis of dsRNAs. The derived virus-related small interfering RNAs (siRNA) mediate cleavage of viral RNAs by post-transcriptional gene silencing (PTGS), thus blocking virus multiplication. Here, we identified ASYMMETRIC LEAVES2 (AS2) as a new component of plant P body complex which mediates mRNA decapping and degradation. We found that AS2 promotes DCP2 decapping activity, accelerates mRNA turnover rate, inhibits siRNA accumulation and functions as an endogenous suppressor of PTGS. Consistent with these findings, as2 mutant plants are resistant to virus infection whereas AS2 over-expression plants are hypersensitive. The geminivirus nuclear shuttle protein BV1 protein, which shuttles between nuclei and cytoplasm, induces AS2 expression, causes nuclear exit of AS2 to activate DCP2 decapping activity and renders infected plants more sensitive to viruses. These principles of gene induction and shuttling of induced proteins to promote mRNA decapping in the cytosol may be used by viral pathogens to weaken antiviral defenses in host plants.