Wusheng Liu

Plant synthetic biology

Plant synthetic biology is an emerging field that combines engineering principles with plant biology toward the design and production of new devices. This emerging field should play an important role in future agriculture for traditional crop improvement, but also in enabling novel bioproduction in plants. In this review we discuss the design cycles of synthetic biology as well as key engineering principles, genetic parts, and computational tools that can be utilized in plant synthetic biology. Some pioneering examples are offered as a demonstration of how synthetic biology can be used to modify plants for specific purposes. These include synthetic sensors, synthetic metabolic pathways, and synthetic genomes. We also speculate about the future of synthetic biology of plants.

Gene expression profiling of resistant and susceptible soybean lines infected with soybean cyst nematode

Soybean cyst nematode (SCN) is the most devastating pathogen of soybean. Information about the molecular basis of soybean–SCN interactions is needed to assist future development of effective management tools against this pathogen. Toward this end, soybean transcript abundance was measured using the Affymetrix Soybean Genome Array in a susceptible and a resistant reaction of soybean to SCN infection. Two genetically related soybean sister lines TN02-226 and TN02-275, which are resistant and susceptible, respectively, to the SCN race 2 infection were utilized in these experiments. Pairwise comparisons followed by false discovery rate analysis indicated that the expression levels of 162 transcripts changed significantly in the resistant line, of which 84 increased while 78 decreased. However, in the susceptible line, 1,694 transcripts changed significantly, of which 674 increased while 1,020 decreased. Comparative analyses of these transcripts indicated that a total of 51 transcripts were in common between resistance and susceptible responses. In this set, 42 transcripts increased in the resistant line, but decreased in the susceptible line. Quantitative real-time reverse-transcription polymerase chain reaction confirmed the results of microarray analysis. Of the transcripts to which a function could be assigned, genes were associated with metabolism, cell wall modification, signal transduction, transcription, and defense. Microarray analyses examining two genetically related soybean lines against the same SCN population provided additional insights into the specific changes in gene expression of a susceptible and a resistant reaction beneficial for identification of genes involved in defense.

Plant synthetic promoters and transcription factors

Synthetic promoters and transcription factors (TFs) have become incredibly powerful and efficient components for precise regulation of targeted plant transgene expression. Synthetic promoters can be rationally designed and constructed using specific type, copy number and spacing of motifs placed upstream of synthetic or native core promoters. Similarly, synthetic TFs can be constructed using a variety of DNA binding domains (DBDs) and effector domains. Synthetic promoters and TFs can provide tremendous advantages over their natural counterparts with regards to transgene expression strength and specificity. They will probably be needed for coordinated transgene expression for metabolic engineering and synthetic circuit applications in plants for bioenergy and advanced crop engineering. In this article we review the recent advances in synthetic promoters and TFs in plants and speculate on their future.

Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing

BackgroundWe aimed to engineer transgenic plants for the purpose of early detection of plant pathogen infection, which was accomplished by employing synthetic pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infection. Toward this end, a number of synthetic promoters consisting of inducible regulatory elements fused to a red fluorescent protein (RFP) reporter were constructed for use in phytosensing.ResultsFor rapid analysis, an Agrobacterium-mediated transient expression assay was evaluated, then utilized to assess the inducibility of each synthetic promoter construct in vivo. Tobacco (Nicotiana tabacum cv. Xanthi) leaves were infiltrated with Agrobacterium harboring the individual synthetic promoter-reporter constructs. The infiltrated tobacco leaves were re-infiltrated with biotic (bacterial pathogens) or abiotic (plant defense signal molecules salicylic acid, ethylene and methyl jasmonate) agents 24 and 48 hours after initial agroinfiltration, followed by RFP measurements at relevant time points after treatment. These analyses indicated that the synthetic promoter constructs were capable of conferring the inducibility of the RFP reporter in response to appropriate phytohormones and bacterial pathogens, accordingly.ConclusionsThese observations demonstrate that the Agrobacterium-mediated transient expression is an efficient method for in vivo assays of promoter constructs in less than one week. Our results provide the opportunity to gain further insights into the versatility of the expression system as a potential tool for high-throughput in planta expression screening prior to generating stably transgenic plants for pathogen phytosensing. This system could also be utilized for temporary phytosensing; e.g., not requiring stably transgenic plants.

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants

Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post-symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early-warning sentinels potentially have tremendous utility as wide-area detectors. We previously showed that synthetic promoters containing pathogen and/or defence signalling inducible cis-acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time-course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.

Computational discovery of soybean promoter cis‐regulatory elements for the construction of soybean cyst nematode‐inducible synthetic promoters

Computational methods offer great hope but limited accuracy in the prediction of functional cis-regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)-inducible motif discovery among promoters of 18 co-expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean-SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co-localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN-inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN-inducible, leading to the discovery of 23 core motifs of 5- to 7-bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W-AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high-throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology.

Field Studies on Dynamic Pollen Production, Deposition, and Dispersion of Glyphosate-Resistant Horseweed (Conyza canadensis)

Abstract Glyphosate-resistant (GR) horseweed has become an especially problematic weed in different crop production systems across the United States and the world. In this field study, we used a nondestructive measurement system to analyze the pollen production, deposition, and dispersion of a Tennessee glyphosate resistant (TNR) horseweed biotype in Knoxville, TN during the 2013 pollination season. We observed that the pollination season of TNR horseweed lasted about 2 mo (54 d). About 78.93% of horseweed pollen was released between 9:00 A.M. and 7:00 P.M. during each sampling day and the release peak was at about 1:30 P.M. The seasonal release of pollen grains was estimated to be 5.11 million grains plant−1. The release rate data indicated that the integrated horizontal flux density and deposition flux density contributed to 78.17% and 21.83% of the release rate, respectively. We also found that pollen concentration decreased with distance from the source field; the average pollen concentration decreased to 50.69% at a distance of 16 m from the source plot. This is the first result of a systematic, direct examination of the release rate (emission and deposition), release pattern (daily and seasonal), and dispersion pattern of GR horseweed pollen. Nomenclature: Glyphosate; horseweed, Conyza canadensis (L.) Cronq ERICA.

Erratum: Advanced genetic tools for plant biotechnology

Nature Reviews Genetics 14, 781–793 (2013) In Figure 4d, e of this article, the products of transgene excision and marker gene removal were shown incorrectly. The article has been corrected online. The authors apologize for the error.