soybean-hairy-root-transformation-for-the-analysis-of-gene-function

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

York University

Glyceollin isomers I, II, and III are the major pathogen-elicited secondary metabolites (i.e. phytoalexins) of soybean () that, collectively with other 5-deoxyisoflavonoids, provide race-specific resistance to  The NAC-family transcription factor (TF) GmNAC42-1 is an essential regulator of some but not all glyceollin biosynthesis genes, indicating other essential TF(s) of the glyceollin gene regulatory network remain to be identified. Here, we conducted comparative transcriptomics on soybean hairy roots of the variety Williams 82 and imbibing seeds of Harosoy 63 upon treatment with wall glucan elicitor from  and identified two homologous R2R3-type MYB TF genes,  and , up-regulated during the times of peak glyceollin biosynthesis. Overexpression and RNA interference silencing of  increased and decreased expression of , , and glyceollin biosynthesis genes and metabolites, respectively, in response to wall glucan elicitor. By contrast, overexpressing or silencing  decreased glyceollin I accumulation with marginal or no effects on the expressions of glyceollin synthesis genes, suggesting a preferential role in promoting glyceollin turnover and/or competing biosynthetic pathways. GmMYB29A2 interacted with the promoters of two glyceollin I biosynthesis genes in vitro and in vivo. Silencing  in Williams 82, a soybean variety that encodes the resistance gene , rendered it compatible with race 1 , whereas overexpressing  rendered the susceptible Williams variety incompatible. Compatibility and incompatibility coincided with reduced and enhanced accumulations of glyceollin I but not other 5-deoxyisoflavonoids. Thus,  is essential for accumulation of glyceollin I and expression of  resistance.

Glyceollin Transcription Factor GmMYB29A2 Regulates Soybean Resistance to .

Cannabis sativa (Cannabis) is a multipurpose plant species consisting of specific lineages that for centuries has either been artificially selected for the production of fiber or the psychoactive drug Δ-tetrahydrocannabinol (THC). With the recent lifting of previous legal restrictions on consuming Cannabis, there has been a resurgence of interest in understanding and manipulating Cannabis genetics to enhance its compositions. Yet, recently developed approaches are not amenable to high-throughput gene stacking to study multi-genic traits. Here, we demonstrate an efficient nanoparticle-based transient gene transformation protocol where multiple gene plasmids can be expressed simultaneously in intact Cannabis leaf cells in a very short time (5 days). Constructs encoding two soybean transcription factors were co-grafted onto poly-ethylenimine cationic polymer-modified silicon dioxide-coated gold nanoparticles (PEI-Au@SiO). Infiltration of the DNA-PEI-Au@SiO into Cannabis leaf tissues resulted in the transcription of both soybean genes and the localization of fluorescent-tagged transcription factor proteins in the nuclei of Cannabis leaf cells including the trichomes, which are the cell types that biosynthesize valuable cannabinoid and terpene metabolites. Our study exemplifies a rapid transient gene transformation approach that will be useful to study the effects of gene stacking in Cannabis.

Nanoparticle-based genetic transformation of Cannabis sativa.

The NAC (NAM, ATAFs, CUC) family of transcription factors (TFs) play a pivotal role in regulating all processes of the growth and development of plants, as well as responses to biotic and abiotic stresses. Yet, the functions of NACs from non-model plant species remains largely uncharacterized. Here, we characterized the stress-responsive effects of a NAC gene isolated from wintersweet, an ornamental woody plant that blooms in winter when temperatures are low. CpNAC68 is clustered in the NAM subfamily. Subcellular localization and transcriptional activity assays demonstrated a nuclear protein that has transcription activator activities. qRT-PCR analyses revealed that  was ubiquitously expressed in old flowers and leaves. Additionally, the expression of  is induced by disparate abiotic stresses and hormone treatments, including drought, heat, cold, salinity, GA, JA, and SA. Ectopic overexpression of  in  enhanced the tolerance of transgenic plants to cold, heat, salinity, and osmotic stress, yet had no effect on growth and development. The survival rate and chlorophyll amounts following stress treatments were significantly higher than wild type Arabidopsis, and were accompanied by lower electrolyte leakage and malondialdehyde (MDA) amounts. In conclusion, our study demonstrates that  can be used as a tool to enhance plant tolerance to multiple stresses, suggesting a role in abiotic stress tolerance in wintersweet.

Transgene  from Wintersweet () Improves Arabidopsis Survival of Multiple Abiotic Stresses.

Quantitative trait loci (QTLs) E and M are major soybean alleles that confer resistance to leaf-chewing insects, and are particularly effective in combination. Flavonoids and/or isoflavonoids are classes of plant secondary metabolites that previous studies agree are the causative agents of resistance of these QTLs. However, all previous studies have compared soybean genotypes that are of dissimilar genetic backgrounds, leaving it questionable what metabolites are a result of the QTL rather than the genetic background. Here, we conducted a non-targeted mass spectrometry approach without liquid chromatography to identify differences in metabolite levels among QTLs E, M, and both (EM) that were introgressed into the background of the susceptible variety Benning. Our results found that E and M mainly confer low-level, global differences in distinct sets of metabolites. The isoflavonoid daidzein was the only metabolite that demonstrated major increases, specifically in insect-treated M and EM. Interestingly, M confers increased daidzein levels in response to insect, whereas E restores M's depleted daidzein levels in the absence of insect. Since daidzein levels do not parallel levels of resistance, our data suggest a novel mechanism that the QTLs confer resistance to insects by mediating changes in hundreds of metabolites, which would be difficult for the insect to evolve tolerance. Collective global metabolite differences conferred by E and M might explain the increased resistance of EM.

Metabolomics Differences of  QTLs Resistant to Soybean Looper.

Glyceollins, isoflavonoid-derived antimicrobial metabolites, are the major phytoalexins in soybean (). They play essential roles in providing resistance to the soil-borne pathogen  and have unconventional anticancer and neuroprotective activities that render them desirable for pharmaceutical development. Our previous studies revealed that the transcription factors GmMYB29A2 and GmNAC42-1 have essential roles in activating glyceollin biosynthesis, yet each cannot activate the transcription of all biosynthesis genes in the absence of a pathogen elicitor treatment. Here, we report that co-overexpressing both transcription factors is also insufficient to activate glyceollin biosynthesis. To understand this insufficiency, we compared the transcriptome profiles of hairy roots overexpressing each transcription factor with glyceollin-synthesizing roots treated with wall glucan elicitor (WGE) from . GmMYB29A2 upregulated most of the WGE-regulated genes that encode enzymatic steps spanning from primary metabolism to the last step of glyceollin biosynthesis. By contrast, GmNAC42-1 upregulated glyceollin biosynthesis genes only when overexpressed in the presence of WGE treatment. This is consistent with our recent discovery that, in the absence of WGE, GmNAC42-1 is bound by GmJAZ1 proteins that inhibit its transactivation activity. WGE, and not GmMYB29A2 or GmNAC42-1, upregulated the heat shock family gene , the homolog of Arabidopsis  that directly activated the transcription of several glyceollin biosynthesis genes. Our results provide important insights into what biosynthesis genes will need to be upregulated to activate the entire glyceollin biosynthetic pathway.

Nik Kovinich

Department of Biology

York University

Soybean Hairy Root Transformation for the Analysis of Gene Function

Nik Kovinich

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

York University

Soybean Hairy Root Transformation for the Analysis of Gene Function

Department of Biology