Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing

Background We 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. Results For 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. Conclusions These 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

A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene

BACKGROUND: Many viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products. RESULTS: We evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus) from cell culture and lymphocystis disease virus (iridovirus) and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1). The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species. CONCLUSION: The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified viruses according to relatedness to sequences of reference viruses and the submission of the sequence data to GenBank will build the database to make the BLAST analysis a valuable resource readily accessible by most diagnostic laboratories. We demonstrated the utility of this assay on viruses that infect fish and birds. These hosts are phylogenetically distant from mammals yet, sequence data suggests that the assay would work equally as well on mammalian counterparts of these groups of viruses. Furthermore, we demonstrated that obtaining genetic information on routine diagnostic samples has great potential for revealing new virus strains and suggesting the presence of new species

Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

Background The expression of fluorescent protein (FP) genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully) appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. Results The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER)-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. Conclusions From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here

Transgenic soybean overexpressing GmSAMT1 exhibits resistance to multiple-HG types of soybean cyst nematode Heterodera glycines

Soybean (Glycine max (L.) Merr.) salicylic acid methyl transferase (GmSAMT1) catalyses the conversion of salicylic acid to methyl salicylate. Prior results showed that when GmSAMT1 was overexpressed in transgenic soybean hairy roots, resistance is conferred against soybean cyst nematode (SCN), Heterodera glycines Ichinohe. In this study, we produced transgenic soybean overexpressing GmSAMT1 and characterized their response to various SCN races. Transgenic plants conferred a significant reduction in the development of SCN HG type 1.2.5.7 (race 2), HG type 0 (race 3) and HG type 2.5.7 (race 5). Among transgenic lines, GmSAMT1 expression in roots was positively associated with SCN resistance. In some transgenic lines, there was a significant decrease in salicylic acid titer relative to control plants. No significant seed yield differences were observed between transgenics and control soybean plants grown in one greenhouse with 22 °C day/night temperature, whereas transgenic soybean had higher yield than controls grown a warmer greenhouse (27 °C day/23 °C night) temperature. In a 1-year field experiment in Knoxville, TN, there was no significant difference in seed yield between the transgenic and nontransgenic soybean under conditions with negligible SCN infection. We hypothesize that GmSAMT1 expression affects salicylic acid biosynthesis, which, in turn, attenuates SCN development, without negative consequences to soybean yield or other morphological traits. Thus, we conclude that GmSAMT1 overexpression confers broad resistance to multiple SCN races, which would be potentially applicable to commercial production

Keeping Data Science Broad: Negotiating the Digital and Data Divide Among Higher Education Institutions

The goal of the “Keeping Data Science Broad” series of webinars and workshops was to garner community input into pathways for keeping data science education broadly inclusive across sectors, institutions, and populations. Input was collected from data science programs across the nation, either traditional or alternative, and from a range of institution types including community colleges, minority-led and minority-serving institutions, liberal arts colleges, tribal colleges, universities, and industry partners. The series consisted of two webinars (August 2017 and September 2017) leading up to a workshop (November 2017) exploring the future of data science education and workforce at institutions of higher learning that are primarily teaching-focused. A third follow-up webinar was held after the workshop (January 2018) to report on outcomes and next steps. Program committee members were chosen to represent a broad spectrum of communities with a diversity of geography (West, Northeast, Midwest, and South), discipline (Computer Science, Math, Statistics, and Domains), as well as institution type (Historically Black Colleges and Universities (HBCU’s), Hispanic-Serving Institutions (HSI’s), other Minority-Serving Institutions (MSI\u27s), Community College\u27s (CC’s), 4-year colleges, Tribal Colleges, R1 Universities, Government and Industry Partners)

Keeping Data Science Broad: Negotiating the Digital and Data Divide Among Higher Education Institutions

The goal of the “Keeping Data Science Broad” series of webinars and workshops was to garner community input into pathways for keeping data science education broadly inclusive across sectors, institutions, and populations. Input was collected from data science programs across the nation, either traditional or alternative, and from a range of institution types including community colleges, minority-led and minority-serving institutions, liberal arts colleges, tribal colleges, universities, and industry partners. The series consisted of two webinars (August 2017 and September 2017) leading up to a workshop (November 2017) exploring the future of data science education and workforce at institutions of higher learning that are primarily teaching-focused. A third follow-up webinar was held after the workshop (January 2018) to report on outcomes and next steps. Program committee members were chosen to represent a broad spectrum of communities with a diversity of geography (West, Northeast, Midwest, and South), discipline (Computer Science, Math, Statistics, and Domains), as well as institution type (Historically Black Colleges and Universities (HBCU’s), Hispanic-Serving Institutions (HSI’s), other Minority-Serving Institutions (MSI\u27s), Community College\u27s (CC’s), 4-year colleges, Tribal Colleges, R1 Universities, Government and Industry Partners)

Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing

<p>Abstract</p> <p>Background</p> <p>We 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.</p> <p>Results</p> <p>For rapid analysis, an <it>Agrobacterium</it>-mediated transient expression assay was evaluated, then utilized to assess the inducibility of each synthetic promoter construct <it>in vivo</it>. Tobacco (<it>Nicotiana tabacum </it>cv. Xanthi) leaves were infiltrated with <it>Agrobacterium </it>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.</p> <p>Conclusions</p> <p>These observations demonstrate that the <it>Agrobacterium</it>-mediated transient expression is an efficient method for <it>in vivo </it>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 <it>in planta </it>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.</p