Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Agroinfiltration for Enhanced Transgene Expression in Coffee Leaves (Coffea arabica L.).

The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation. This chapter describes an ex vitro protocol for transient expression using A. tumefaciens-mediated infiltration of coffee leaves, which could be used to produce coffee plants expressing desirable traits against biotic and abiotic stresses, genes controlling biochemical and physiological traits, as well as for gene editing through CRISPR/Cas9.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

Insight in the quorum sensing-driven lifestyle of the non-pathogenic Agrobacterium tumefaciens 6N2 and the interactions with the yeast Meyerozyma guilliermondii.

Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the QS regulation in the bacteria-yeast interactions.

A Sinorhizobium meliloti and Agrobacterium tumefaciens ExoR ortholog is not crucial for Brucella abortus virulence.

Brucella is a facultative extracellular-intracellular pathogen that belongs to the Alphaproteobacteria class. Precise sensing of environmental changes and a proper response mediated by a gene expression regulatory network are essential for this pathogen to survive. The plant-related Alphaproteobacteria Sinorhizobium meliloti and Agrobacterium tumefaciens also alternate from a free to a host-associated life, where a regulatory invasion switch is needed for this transition. This switch is composed of a two-component regulatory system (TCS) and a global inhibitor, ExoR. In B. abortus, the BvrR/BvrS TCS is essential for intracellular survival. However, the presence of a TCS inhibitor, such as ExoR, in Brucella is still unknown. In this work, we identified a genomic sequence similar to S. meliloti exoR in the B. abortus 2308W genome, constructed an exoR mutant strain, and performed its characterization through ex vivo and in vivo assays. Our findings indicate that ExoR is related to the BvrR phosphorylation state, and is related to the expression of known BvrR/BrvS gene targets, such as virB8, vjbR, and omp25 when grown in rich medium or starving conditions. Despite this, the exoR mutant strain showed no significant differences as compared to the wild-type strain, related to resistance to polymyxin B or human non-immune serum, intracellular replication, or infectivity in a mice model. ExoR in B. abortus is related to BvrR/BvrS as observed in other Rhizobiales; however, its function seems different from that observed for its orthologs described in A. tumefaciens and S. meliloti.

Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods.

MAIN CONCLUSION: The combined Agrobacterium- and biolistic-mediated methods of cotton transformation provide a straightforward and highly efficient protocol for obtaining transgenic cotton. Cotton (Gossypium spp.) is the most important crop for natural textile fiber production worldwide. Nonetheless, one of the main challenges in cotton production are the losses resulting from insect pests, pathogens, and abiotic stresses. One effective way to solve these issues is to use genetically modified (GM) varieties. Herein, we describe an improved protocol for straightforward and cost-effective genetic transformation of cotton embryo axes, merging biolistics and Agrobacterium. The experimental steps include (1) Agrobacterium preparation, (2) seed sterilization, (3) cotton embryo excision, (4) lesion of shoot-cells by tungsten bombardment, (5) Agrobacterium-mediated transformation, (6) embryo co-culture, (7) regeneration and selection of transgenic plants in vitro, and (8) molecular characterization of plants. Due to the high regenerative power of the embryonic axis and the exceptional ability of the meristem cells for plant regeneration through organogenesis in vitro, this protocol can be performed in approximately 4-10 weeks, with an average plant regeneration of about 5.5% (± 0.53) and final average transformation efficiency of 60% (± 0.55). The transgene was stably inherited, and most transgenic plants hold a single copy of the transgene, as desirable and expected in Agrobacterium-mediated transformation. Additionally, the transgene was stably expressed over generations, and transgenic proteins could be detected at high levels in the T2 generation of GM cotton plants. The T2 progeny showed no phenotypic or productivity disparity compared to wild-type plants. Collectively, the use of cotton embryo axes and the enhanced DNA-delivery system by combining particle bombardment and Agrobacterium infection enabled efficient transgenic plant recovery, overcoming usual limitations associated with the recalcitrance of several cotton genotypes subjected to somatic embryogenesis. The improved approach states this method's success for cotton genetic modification, allowing us to obtain GM cotton plants carrying traits, which are of fundamental relevance for the advancement of global agribusiness.

Visualization of Transiently Expressed mRNA in Plants Using MS2.

RNA transport and localization are evolutionarily conserved processes that allow protein translation to occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates, embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA transport to establish localized replication centers within infected cells and then to disseminate their infectious genomes between cells and throughout the plant organism with the help of their movement proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a 19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent protein-tagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA imaging data.

An improved protocol for efficient transformation and regeneration of Setaria italica.

KEY MESSAGE: An efficient and improved transformation method for functional genetics studies in S. italica, being a boon for the Setaria scientific community and for crop improvement. Foxtail millet (Setaria italica) is a short life cycle C4 plant, with sequenced genome, and a potential model plant for C4 species. S. italica is also important on a global food security and healthiness context due to its importance in arid and semi-arid areas. However, despite its importance, there are just few transformation protocols directed to this species. The current protocols reached about 5.5-9% of efficiency, which do not make it a valuable model organism. Different types of explants were used in the above mentioned methods, such as immature and mature inflorescence and shoot apex. However, these techniques have many limitations, such as unavailability of explants throughout the year and a crucial, laborious and considerable time-consuming selection. Aiming a simplified and efficient methodology, we adopted dry mature seeds as explants, which are available in abundance, are constant along the year and well responsive to tissue culture, in addition to a differentiated approach that reaches on an average 19.2% transformation efficiency of S. italica. Thus, we propose a protocol that optimizes the transformation efficiency of this cereal crop allowing a high increase on transformation and regeneration rates. Our transformation protocol provides an interesting tool for Setaria community research as well as enables new strategies for breeding enhanced productivity in the species.

Agrobacterium tumefaciens-Mediated Stable Transformation of Daucus carota.

Daucus carota L. (carrot) is one of the ten most important vegetables cultivated and consumed worldwide and is a main source of provitamin A. Carrot storage root is rich in dietary fiber, antioxidants, and other nutrients but especially in carotenoids. It has been also used as plant model for studding embryogenesis, as well as the genetic and genomic evolution of carrots and for carotenoid synthesis regulation, among others. Research in carrot often needs genetic transformation. Here we describe a step-by-step protocol on the nuclear and stable transformation of carrot through Agrobacterium tumefaciens and somatic embryogenesis in vitro culture. Somatic embryos, induced by supplementation of Murashige-Skoog medium with the 2,4D hormone, develop into seedlings after 6 months approximately when plants are ready to be transferred to a greenhouse. The protocol has over 85% of transformation efficiency.

Insight into the structure, function and conjugative transfer of pLPU83a, an accessory plasmid of Rhizobium favelukesii LPU83.

Plasmids are widely distributed in rhizobia, a group of bacteria able to establish symbiotic relationships with the roots of legume plants. Two types of conjugative transfer (CT) regulation of these elements have been described in more detail. The most prevalent is through Quorum-Sensing (QS), mediated by the interaction of the TraR regulator protein and its cognate acyl-homoserine lactone (AHL) synthesized by TraI. In this study, we analyzed rhizobial plasmids classified according to their TraR regulators into four different groups. Each group has a particular genomic architecture. In one of the groups (I-C), represented by pLPU83a from Rhizobium favelukesii LPU83, CT induction requires TraR. With manual annotation, a traI was located in the plasmid distant to the traR gene. These features make pLPU83a an interesting plasmid for studying novel mechanisms of CT regulation. We mutagenized the traI gene, and found that it does not participate in CT regulation. Furthermore, we studied whether pLPU83a is subject to QS regulation by determining CT at different growth stages (cell densities). Our results showed no positive correlation between increase in culture densities and CT induction, on the contrary a slight decrease in CT was found at higher culture densities, unlike other TraR-depending plasmids. Our results show that transfer of pLPU83a is not regulated in a QS-dependent manner, and suggest that molecules not yet identified may activate its CT. Also, accumulation of a putative inhibitor cannot be disregarded.

New approach for the construction of infectious clones of a circular DNA plant virus using Gibson Assembly.

Viruses belonging to the genus Begomovirus (family Geminiviridae) have circular single-strand DNA genomes encapsidated into quasi-icosahedral particles, and are transmitted by whiteflies of the Bemisia tabaci complex. Biological and molecular properties of begomoviruses have been studied efficiently with infectious clones containing dimeric genomic components. However, current approaches employing enzymatic digestion and ligation to binary vectors are laborious, mostly due to many cloning steps or partial digestion by restriction enzyme. Here, an infectious clone of the bipartite begomovirus Bean golden mosaic virus (BGMV) was obtained using PCR and Gibson Assembly (GA). Common bean (Phaseolus vulgaris) seedlings displayed severe yellow mosaic and stunt symptoms 15 days after agroinoculation with DNA-A and DNA-B of BGMV. The approach based on PCR-GA protocol is a fast and useful tool to obtain infectious clones of a circular DNA plant virus.

Setaria viridis as a Model Plant for Functional Genomic Studies in C4 Crops.

Setaria viridis is an emerging model for C4 species, and it is an important model to validate some genes for further C4 crop transformation, such as sugarcane, maize, and wheat. Here, we describe two protocols for stable transformation of S. viridis mediated by Agrobacterium tumefaciens with three different reporter genes and two selectable markers. Routine transformation efficiency reaching 29% was achieved using embryogenic callus in S. viridis (accession A10.1). Alternatively, we developed a transformation method by floral dip with 0.6% efficiency. The developed protocols could be useful for genetic and genomics studies of important food-feed-fiber-fuel C4 crops.

Generation of a synthetic binary plasmid that confers resistance to nourseothricin for genetic engineering of Sporothrix schenckii.

Some members of the Sporothrix genus can cause sporotrichosis, a worldwide distributed mycosis that affects several mammalian species, including human beings. Sporothrix schenckii and Sporothrix brasiliensis are the fungal species frequently associated with this disease, and the latter has gained significant interest because of the increased number of cases associated with transmission by cats. Despite the relevance of these organisms in the medical field, limited strategies for their genetic manipulation have been explored. Thus far, gene silencing using the hygromycin B resistance cassette is the sole strategy currently available to study these organisms. Here, we report the generation of a cassette that confers resistance to nourseothricin, which was successfully transferred from Agrobacterium tumefaciens to Sporothrix cells. Therefore, this can be used as a second selective marker to manipulate the genome of these organisms.

Generation of Sporothrix schenckii mutants expressing the green fluorescent protein suitable for the study of host-fungus interactions.

Sporotrichosis is an infection caused by members of the Sporothrix genus, and among them, Sporothrix schenckii is one of the etiological agents. Both, the disease and the causative agent have gained interest in the recent years, because of the report of epidemic outbreaks, and the description of the disease transmission from animals to human beings. Despite the relevance of S. schenckii in the clinical field, there are basic aspects of its biology poorly explored. So far, Agrobacterium tumefaciens-mediated transformation has been reported as an alternative for genetic manipulation of this fungal pathogen. Here, we report the optimization of the transformation method and used this to generate insertional mutants that express the green fluorescent protein in S. schenckii. We obtained five mutant strains that showed mitotic stability and expression of the reporter gene. The strains displayed normal cell wall composition, and a similar ability to interact ex vivo with human monocytes and monocyte-derived macrophages. Moreover, the virulence in larvae of Galleria mellonella was similar to that obtained with the wild-type control strains. These data indicate that these fluorescent mutants with normal ability to interact with the host could be used in bioimaging to track the host-Sporothrix interaction in vivo.

Vacuolar Targeting and Characterization of Recombinant Antibodies.

Plant-based platforms are extensively use for the expression of recombinant proteins, including monoclonal antibodies (mAbs). Generally, immunoglobulins (Igs) are sorted to the apoplast, which is often afflicted with intense proteolysis. Here, we describe methods to transiently express mAbs sorted to central vacuole in Nicotiana benthamiana leaves and to characterize the obtained IgG. Central vacuole is an appropriate compartment for the efficient production of Abs, consequently vacuolar sorting should be considered as an alternative strategy to obtain high protein yields.

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD600 = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.

BigR is a sulfide sensor that regulates a sulfur transferase/dioxygenase required for aerobic respiration of plant bacteria under sulfide stress.

To cope with toxic levels of H2S, the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens employ the bigR operon to oxidize H2S into sulfite. The bigR operon is regulated by the transcriptional repressor BigR and it encodes a bifunctional sulfur transferase (ST) and sulfur dioxygenase (SDO) enzyme, Blh, required for H2S oxidation and bacterial growth under hypoxia. However, how Blh operates to enhance bacterial survival under hypoxia and how BigR is deactivated to derepress operon transcription is unknown. Here, we show that the ST and SDO activities of Blh are in vitro coupled and necessary to oxidize sulfide into sulfite, and that Blh is critical to maintain the oxygen flux during A. tumefaciens respiration when oxygen becomes limited to cells. We also show that H2S and polysulfides inactivate BigR leading to operon transcription. Moreover, we show that sulfite, which is produced by Blh in the ST and SDO reactions, is toxic to Citrus sinensis and that X. fastidiosa-infected plants accumulate sulfite and higher transcript levels of sulfite detoxification enzymes, suggesting that they are under sulfite stress. These results indicate that BigR acts as a sulfide sensor in the H2S oxidation mechanism that allows pathogens to colonize plant tissues where oxygen is a limiting factor.

Genetic transformation of the Brazilian BR 451 maize variety by the Agrobacterium tumefaciens method / Transformação genética da variedade brasileira de milho BR 451 pelo método da Agrobacterium tumefaciens

The asexually gene introduction by genetic engineering has brought enormous possibilities to innovate plant breeding. However, principally because of the low in vitro response, genetic transformation has been restricted to only certain genotypes of agronomically significant species. With the objective of establishing a protocol for genetically transforming the Brazilian BR 451 maize variety through Agrobacterium tumefaciens, it was studied the capacity of plant regeneration in vitro from embryogenic calli cultivated in three regeneration media, each having different growth regulators. It was also evaluated the temperature stress effect on the transformation of the immature embryos with A. tumefaciens EHA 101 containing the plasmid pTF102 with uidA and bar genes. The BR 451 variety embryos and those of the Hi-II hybrid (control) were exposed to three treatments applied as they were being infected with the agrobacteria (a) infection at 25°C; (b) infection at 40°C; (c) pretreatment at 40°C for 5 seconds followed by infection at 25°C. Transformation was determined by uidA gene expression and through the callus resistant to the herbicide Bialaphos® formation. Embryos infected at 40°C showed a higher degree of genetic transformation in the Hi-II, although the same was not noted in BR 451. When growth regulators were added to the culture medium the number of regenerated BR 451 plants showed no increase.(AU)

A introdução de genes de forma assexual por meio da engenharia genética tem ampliado as possibilidades do melhoramento genético vegetal. No entanto, devido principalmente a baixa resposta in vitro, a transformação genética tem se limitado a poucos genótipos das espécies de interesse agronômico. Visando estabelecer protocolo de transformação genética da variedade de milho BR 451 via Agrobacterium tumefaciens, foi estudada a capacidade de regeneração de plantas in vitro a partir de calos embriogênicos cultivados em três meios de regeneração contendo diferentes reguladores de crescimento. Também foi avaliado o efeito do estresse de temperatura na transformação de embriões imaturos com a A. tumefaciens EHA 101portadora do plasmídeo pTF102 que contém os genes uidA e bar. Para tal, três tratamentos foram aplicados aos embriões da variedade BR 451 e do híbrido Hi-II (controle) durante a infecção com a agrobactéria: (a) infecção em 25oC; (b) infecção a 40oC; (c) pré-tratamento de 40oC por cinco segundos seguido por infecção em 25oC. A transformação foi avaliada mediante a expressão do gene uidA e a formação de calos resistentes ao herbicida Bialaphos®. A infecção de embriões a 40oC aumentou a transformação genética em Hi-II, mas não em BR 451. A adição de reguladores de crescimento no meio de regeneração não incrementou o número de plantas regeneradas.(AU)

Increased Postharvest Life of TomLox B Silenced Mutants of Tomato (Solanum lycopersicum) Var. TA234.

A healthy lifestyle includes fruits and vegetables consumption. Tomato is one of the most consumed vegetables, although it is susceptible to physical damage through postharvest handling, thus leading to important losses. Softening is an important variable during tomato ripening; excessive softening is undesirable and leads to postharvest losses. TomloxB plays an important role in ripening, mainly in the loss of cellular integrity caused by fatty acids released from the lipid matrix of membranes that initiate oxidative deterioration, which is in turn carried into senescence. In order to increase postharvest life, we produced transgenic tomato plants via Rhizobium radiobacter with tomato lipoxygenase B (TomloxB) antisense constructs under control of the cauliflower mosaic virus (CaMV) 35S promoter. Lipoxygenase activity and firmness were measured in tomato fruit and the fatty acids profile was determined. Transgenic fruits were maintained for 40 days at room temperature in optimal conditions, whereas wild type fruits remained in similar conditions for only six days. Firmness in pink and red stages was significantly lower in wild type fruits than in two transgenic lines. Linolenic acid was the most important fatty acid consumed by lipoxygenase in both turning and pink stages of ripening. Lipoxygenase activity was smaller in transformed fruits in comparison with the wild type. These results suggest that silencing the TomloxB gene promoted significant changes in the physiology of transformed tomatoes, being the increase in postharvest life the most important.