A comprehensive review of in planta stable transformation strategies.

Plant transformation remains a major bottleneck to the improvement of plant science, both on fundamental and practical levels. The recalcitrant nature of most commercial and minor crops to genetic transformation slows scientific progress for a large range of crops that are essential for food security on a global scale. Over the years, novel stable transformation strategies loosely grouped under the term "in planta" have been proposed and validated in a large number of model (e.g. Arabidopsis and rice), major (e.g. wheat and soybean) and minor (e.g. chickpea and lablab bean) species. The in planta approach is revolutionary as it is considered genotype-independent, technically simple (i.e. devoid of or with minimal tissue culture steps), affordable, and easy to implement in a broad range of experimental settings. In this article, we reviewed and categorized over 300 research articles, patents, theses, and videos demonstrating the applicability of different in planta transformation strategies in 105 different genera across 139 plant species. To support this review process, we propose a classification system for the in planta techniques based on five categories and a new nomenclature for more than 30 different in planta techniques. In complement to this, we clarified some grey areas regarding the in planta conceptual framework and provided insights regarding the past, current, and future scientific impacts of these techniques. To support the diffusion of this concept across the community, this review article will serve as an introductory point for an online compendium about in planta transformation strategies that will be available to all scientists. By expanding our knowledge about in planta transformation, we can find innovative approaches to unlock the full potential of plants, support the growth of scientific knowledge, and stimulate an equitable development of plant research in all countries and institutions.

Dissection of the E8 locus in two early maturing Canadian soybean populations.

Soybean [Glycine max (L.) Merr.] is a short-day crop for which breeders want to expand the cultivation range to more northern agro-environments by introgressing alleles involved in early reproductive traits. To do so, we investigated quantitative trait loci (QTL) and expression quantitative trait loci (eQTL) regions comprised within the E8 locus, a large undeciphered region (~7.0 Mbp to 44.5 Mbp) associated with early maturity located on chromosome GM04. We used a combination of two mapping algorithms, (i) inclusive composite interval mapping (ICIM) and (ii) genome-wide composite interval mapping (GCIM), to identify major and minor regions in two soybean populations (QS15524F2:F3 and QS15544RIL) having fixed E1, E2, E3, and E4 alleles. Using this approach, we identified three main QTL regions with high logarithm of the odds (LODs), phenotypic variation explained (PVE), and additive effects for maturity and pod-filling within the E8 region: GM04:16,974,874-17,152,230 (E8-r1); GM04:35,168,111-37,664,017 (E8-r2); and GM04:41,808,599-42,376,237 (E8-r3). Using a five-step variant analysis pipeline, we identified Protein far-red elongated hypocotyl 3 (Glyma.04G124300; E8-r1), E1-like-a (Glyma.04G156400; E8-r2), Light-harvesting chlorophyll-protein complex I subunit A4 (Glyma.04G167900; E8-r3), and Cycling dof factor 3 (Glyma.04G168300; E8-r3) as the most promising candidate genes for these regions. A combinatorial eQTL mapping approach identified significant regulatory interactions for 13 expression traits (e-traits), including Glyma.04G050200 (Early flowering 3/E6 locus), with the E8-r3 region. Four other important QTL regions close to or encompassing major flowering genes were also detected on chromosomes GM07, GM08, and GM16. In GM07:5,256,305-5,404,971, a missense polymorphism was detected in the candidate gene Glyma.07G058200 (Protein suppressor of PHYA-105). These findings demonstrate that the locus known as E8 is regulated by at least three distinct genomic regions, all of which comprise major flowering genes.

The SoyaGen Project: Putting Genomics to Work for Soybean Breeders.

The SoyaGen project was a collaborative endeavor involving Canadian soybean researchers and breeders from academia and the private sector as well as international collaborators. Its aims were to develop genomics-derived solutions to real-world challenges faced by breeders. Based on the needs expressed by the stakeholders, the research efforts were focused on maximizing realized yield through optimization of maturity and improved disease resistance. The main deliverables related to molecular breeding in soybean will be reviewed here. These include: (1) SNP datasets capturing the genetic diversity within cultivated soybean (both within a worldwide collection of > 1,000 soybean accessions and a subset of 102 short-season accessions (MG0 and earlier) directly relevant to this group); (2) SNP markers for selecting favorable alleles at key maturity genes as well as loci associated with increased resistance to key pathogens and pests (Phytophthora sojae, Heterodera glycines, Sclerotinia sclerotiorum); (3) diagnostic tools to facilitate the identification and mapping of specific pathotypes of P. sojae; and (4) a genomic prediction approach to identify the most promising combinations of parents. As a result of this fruitful collaboration, breeders have gained new tools and approaches to implement molecular, genomics-informed breeding strategies. We believe these tools and approaches are broadly applicable to soybean breeding efforts around the world.

Identification of novel loci associated with maturity and yield traits in early maturity soybean plant introduction lines.

BACKGROUND: To continue to meet the increasing demands of soybean worldwide, it is crucial to identify key genes regulating flowering and maturity to expand the cultivated regions into short season areas. Although four soybean genes have been successfully utilized in early maturity breeding programs, new genes governing maturity are continuously being identified suggesting that there remains as yet undiscovered loci governing agronomic traits of interest. The objective of this study was to identify novel loci and genes involved in a diverse set of early soybean maturity using genome-wide association (GWA) analyses to identify loci governing days to maturity (DTM), flowering (DTF) and pod filling (DTPF), as well as yield and 100 seed weight in Canadian environments. To do so, soybean plant introduction lines varying significantly for maturity, but classified as early varieties, were used. Plants were phenotyped for the five agronomic traits for five site-years and GWA approaches used to identify candidate loci and genes affecting each trait. RESULTS: Genotyping using genotyping-by-sequencing and microarray methods identified 67,594 single nucleotide polymorphisms, of which 31,283 had a linkage disequilibrium < 1 and minor allele frequency > 0.05 and were used for GWA analyses. A total of 9, 6, 4, 5 and 2 loci were detected for GWA analyses for DTM, DTF, DTPF, 100 seed weight and yield, respectively. Regions of interest, including a region surrounding the E1 gene for flowering and maturity, and several novel loci, were identified, with several loci having pleiotropic effects. Novel loci affecting maturity were identified on chromosomes five and 13 and reduced maturity by 7.2 and 3.3 days, respectively. Novel loci for maturity and flowering contained genes orthologous to known Arabidopsis flowering genes, while loci affecting yield and 100 seed weight contained genes known to cause dwarfism. CONCLUSIONS: This study demonstrated substantial variation in soybean agronomic traits of interest, including maturity and flowering dates as well as yield, and the utility of GWA analyses in identifying novel genetic factors underlying important agronomic traits. The loci and candidate genes identified serve as promising targets for future studies examining the mechanisms underlying the related soybean traits.

Antioxidant genes of plants and fungal pathogens are distinctly regulated during disease development in different Rhizoctonia solani pathosystems.

Biotic stress, as a result of plant-pathogen interactions, induces the accumulation of reactive oxygen species in the cells, causing severe oxidative damage to plants and pathogens. To overcome this damage, both the host and pathogen have developed antioxidant systems to quench excess ROS and keep ROS production and scavenging systems under control. Data on ROS-scavenging systems in the necrotrophic plant pathogen Rhizoctonia solani are just emerging. We formerly identified vitamin B6 biosynthetic machinery of R. solani AG3 as a powerful antioxidant exhibiting a high ability to quench ROS, similar to CATALASE (CAT) and GLUTATHIONE S-TRANSFERASE (GST). Here, we provide evidence on the involvement of R. solani vitamin B6 biosynthetic pathway genes; RsolPDX1 (KF620111.1), RsolPDX2 (KF620112.1), and RsolPLR (KJ395592.1) in vitamin B6 de novo biosynthesis by yeast complementation assays. Since gene expression studies focusing on oxidative stress responses of both the plant and the pathogen following R. solani infection are very limited, this study is the first coexpression analysis of genes encoding vitamin B6, CAT and GST in plant and fungal tissues of three pathosystems during interaction of different AG groups of R. solani with their respective hosts. The findings indicate that distinct expression patterns of fungal and host antioxidant genes were correlated in necrotic tissues and their surrounding areas in each of the three R. solani pathosystems: potato sprout-R. solani AG3; soybean hypocotyl-R. solani AG4 and soybean leaves-R. solani AG1-IA interactions. Levels of ROS increased in all types of potato and soybean tissues, and in fungal hyphae following infection of R. solani AGs as determined by non-fluorescence and fluorescence methods using H2DCF-DA and DAB, respectively. Overall, we demonstrate that the co-expression and accumulation of certain plant and pathogen ROS-antioxidant related genes in each pathosystem are highlighted and might be critical during disease development from the plant's point of view, and in pathogenicity and developing of infection structures from the fungal point of view.

The transcriptional landscape of Rhizoctonia solani AG1-IA during infection of soybean as defined by RNA-seq.

Rhizoctonia solani Kühn infects most plant families and can cause significant agricultural yield losses worldwide; however, plant resistance to this disease is rare and short-lived, and therefore poorly understood, resulting in the use of chemical pesticides for its control. Understanding the functional responses of this pathogen during host infection can help elucidate the molecular mechanisms that are necessary for successful host invasion. Using the pathosystem model soybean-R. solani anastomosis group AG1-IA, we examined the global transcriptional responses of R. solani during early and late infection stages of soybean by applying an RNA-seq approach. Approximately, 148 million clean paired-end reads, representing 93% of R. solani AG1-IA genes, were obtained from the sequenced libraries. Analysis of R. solani AG1-IA transcripts during soybean invasion revealed that most genes were similarly expressed during early and late infection stages, and only 11% and 15% of the expressed genes were differentially expressed during early and late infection stages, respectively. Analyses of the differentially expressed genes (DEGs) revealed shifts in molecular pathways involved in antibiotics biosynthesis, amino acid and carbohydrate metabolism, as well as pathways involved in antioxidant production. Furthermore, several KEGG pathways were unique to each time point, particularly the up-regulation of genes related to toxin degradation (e.g., nicotinate and nicotinamid metabolism) at onset of necrosis, and those linked to synthesis of anti-microbial compounds and pyridoxine (vitamin B6) biosynthesis 24 h.p.o. of necrosis. These results suggest that particular genes or pathways are required for either invasion or disease development. Overall, this study provides the first insights into R. solani AG1-IA transcriptome responses to soybean invasion providing beneficial information for future targeted control methods of this successful pathogen.

An integrated RNAseq-1H NMR metabolomics approach to understand soybean primary metabolism regulation in response to Rhizoctonia foliar blight disease.

BACKGROUND: Rhizoctonia solani AG1-IA is a devastating phytopathogen causing Rhizoctonia foliar blight (RFB) of soybean worldwide with yield losses reaching 60%. Plant defense mechanisms are complex and information from different metabolic pathways is required to thoroughly understand plant defense regulation and function. Combining information from different "omics" levels such as transcriptomics, metabolomics, and proteomics is required to gain insights into plant metabolism and its regulation. As such, we studied fluctuations in soybean metabolism in response to R. solani infection at early and late disease stages using an integrated transcriptomics-metabolomics approach, focusing on the regulation of soybean primary metabolism and oxidative stress tolerance. RESULTS: Transcriptomics (RNAseq) and metabolomics (1H NMR) data were analyzed individually and by integration using bidirectional orthogonal projections to latent structures (O2PLS) to reveal possible links between the metabolome and transcriptome during early and late infection stages. O2PLS analysis detected 516 significant transcripts, double that reported in the univariate analysis, and more significant metabolites than detected in partial least squares discriminant analysis. Strong separation of treatments based on integration of the metabolomes and transcriptomes of the analyzed soybean leaves was revealed, similar trends as those seen in analyses done on individual datasets, validating the integration method being applied. Strong fluctuations of soybean primary metabolism occurred in glycolysis, the TCA cycle, photosynthesis and photosynthates in response to R. solani infection. Data were validated using quantitative real-time PCR on a set of specific markers as well as randomly selected genes. Significant increases in transcript and metabolite levels involved in redox reactions and ROS signaling, such as peroxidases, thiamine, tocopherol, proline, L-alanine and GABA were also recorded. Levels of ethanol increased 24 h post-infection in soybean leaves, and alcohol dehydrogenase (ADH) loss-of-function mutants of Arabidopsis thaliana had higher necrosis than wild type plants. CONCLUSIONS: As a proof-of-concept, this study offers novel insights into the biological correlations and identification of candidate genes and metabolites that can be used in soybean breeding for resistance to R. solani AG1-IA infection. Additionally, these findings imply that alcohol and its associated gene product ADH may have important roles in plant resistance to R. solani AG1-IA causing foliar blight.

Biochar Amendment Modifies Expression of Soybean and Rhizoctonia solani Genes Leading to Increased Severity of Rhizoctonia Foliar Blight.

Application of biochar, a pyrolyzed biomass from organic sources, to agricultural soils is considered a promising strategy to sustain soil fertility leading to increased plant productivity. It is also known that applications of biochar to soilless potting substrates and to soil increases resistance of plants against diseases, but also bear the potential to have inconsistent and contradictory results depending on the type of biochar feedstock and application rate. The following study examined the effect of biochar produced from maple bark on soybean resistance against Rhizoctonia foliar blight (RFB) disease caused by Rhizoctonia solani, and examined the underlying molecular responses of both soybean and R. solani during interaction with biochar application. Soybean plants were grown in the presence of 1, 3, or 5% (w/w) or absence of maple bark biochar for 2 weeks, and leaves were infected with R. solani AG1-IA. At lower concentrations (1 and 3%), biochar was ineffective against RFB, however at the 5% amendment rate, biochar was conducive to RFB with a significant increase in disease severity. For the first time, soybean and R. solani responsive genes were monitored during the development of RFB on detached leaves of plants grown in the absence and presence of 5% biochar at 0, 6, 12, and 24 h post-inoculation (h.p.i.). Generally, large decreases in soybean transcript abundances of genes associated with primary metabolism such as glycolysis, tricarboxylic acid (TCA) cycle, starch, amino acid and glutathione metabolism together with genes associated with plant defense and immunity such as salicylic acid (SA) and jasmonic acid pathways were observed after exposure of soybean to high concentration of biochar. Such genes are critical for plant protection against biotic and abiotic stresses. The general down-regulation of soybean genes and changes in SA hormonal balance were tightly linked with an increased susceptibility to RFB. In conjunction, R. solani genes associated with carbohydrate metabolism were up-regulated, while genes involved in redox reactions and detoxification had varying effects. In conclusion, this study presents strong evidence that maple bark biochar increased susceptibility of soybean to a foliar disease. This condition is partly mediated by the down-regulation of soybean genes leading to reduced immunity and also affecting R. solani gene expression.

Maple Bark Biochar Affects Rhizoctonia solani Metabolism and Increases Damping-Off Severity.

Many studies have investigated the effect of biochar on plant yield, nutrient uptake, and soil microbial populations; however, little work has been done on its effect on soilborne plant diseases. To determine the effect of maple bark biochar on Rhizoctonia damping-off, 11 plant species were grown in a soilless potting substrate amended with different concentrations of biochar and inoculated or not with Rhizoctonia solani anastomosis group 4. Additionally, the effect of biochar amendment on R. solani growth and metabolism in vitro was evaluated. Increasing concentrations of maple bark biochar increased Rhizoctonia damping-off of all 11 plant species. Using multivariate analyses, we observed positive correlations between biochar amendments, disease severity and incidence, abundance of culturable bacterial communities, and physicochemical parameters. Additionally, biochar amendment significantly increased R. solani growth and hyphal extension in vitro, and altered its primary metabolism, notably the mannitol and tricarboxylic acid cycles and the glycolysis pathway. One or several organic compounds present in the biochar, as identified by gas chromatography-mass spectrometry analysis, may be metabolized by R. solani. Taken together, these results indicate that future studies on biochar should focus on the effect of its use as an amendment on soilborne plant pathogens before applying it to soils.

Arabidopsis redox status in response to caterpillar herbivory.

Plant responses to insect herbivory are regulated through complex, hormone-mediated interactions. Some caterpillar species have evolved strategies to manipulate this system by inducing specific pathways that suppress plant defense responses. Effectors in the labial saliva (LS) secretions of Spodoptera exigua caterpillars are believed to induce the salicylic acid (SA) pathway to interfere with the jasmonic acid (JA) defense pathway; however, the mechanism underlying this subversion is unknown. Since noctuid caterpillar LS contains enzymes that may affect cellular redox balance, this study investigated rapid changes in cellular redox metabolites within 45 min after herbivory. Caterpillar LS is involved in suppressing the increase in oxidative stress that was observed in plants fed upon by caterpillars with impaired LS secretions. To further understand the link between cellular redox balance and plant defense responses, marker genes of SA, JA and ethylene (ET) pathways were compared in wildtype, the glutathione-compromised pad2-1 mutant and the tga2/5/6 triple mutant plants. AtPR1 and AtPDF1.2 showed LS-dependent expression that was alleviated in the pad2-1 and tga2/5/6 triple mutants. In comparison, the ET-dependent genes ERF1 expression showed LS-associated changes in both wildtype and pad2-1 mutant plants and the ORA 59 marker AtHEL had increased expression in response to herbivory, but a LS-dependent difference was not noted. These data support the model that there are SA/NPR1-, glutathione-dependent and ET-, glutathione-independent mechanisms leading to LS-associated suppression of plant induced defenses.

Gas chromatography-mass spectrometry metabolite profiling of worker honey bee (Apis mellifera L.) hemolymph for the study of Nosema ceranae infection.

Here, we are presenting a gas chromatography-mass spectrometry (GC/MS) approach for the study of infection of the worker honey bee (Apis mellifera L.) by the newly emerged obligate intracellular parasite Nosema ceranae based on metabolite profiling of hemolymph. Because of the severity of the disease, early detection is crucial for its efficient control. Results revealed that the parasite causes a general disturbance of the physiology of the honey bee affecting the mechanisms controlling the mobilization of energy reserves in infected individuals. The imposed nutritional and energetic stress to the host was depicted mainly in the decreased levels of the majority of carbohydrates and amino acids, including metabolites such as fructose, l-proline, and the cryoprotectants sorbitol and glycerol, which are implicated in various biochemical pathways. Interestingly, the level of glucose was detected at significantly higher levels in infected honey bees. Metabolomics analyses were in agreement with those of multiplex quantitative PCR analyses, indicating that it can be used as a complementary tool for the detection and the study of the physiology of the disease.