Editing of SlWRKY29 by CRISPR-activation promotes somatic embryogenesis in Solanum lycopersicum cv. Micro-Tom.

At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.

The Cytokinins BAP and 2-iP Modulate Different Molecular Mechanisms on Shoot Proliferation and Root Development in Lemongrass (Cymbopogon citratus).

The known activities of cytokinins (CKs) are promoting shoot multiplication, root growth inhibition, and delaying senescence. 6-Benzylaminopurine (BAP) has been the most effective CK to induce shoot proliferation in cereal and grasses. Previously, we reported that in lemongrass (Cymbopogon citratus) micropropagation, BAP 10 µM induces high shoot proliferation, while the natural CK 6-(γ,γ-Dimethylallylamino)purine (2-iP) 10 µM shows less pronounced effects and developed rooting. To understand the molecular mechanisms involved, we perform a protein-protein interaction (PPI) network based on the genes of Brachypodium distachyon involved in shoot proliferation/repression, cell cycle, stem cell maintenance, auxin response factors, and CK signaling to analyze the molecular mechanisms in BAP versus 2-iP plants. A different pattern of gene expression was observed between BAP- versus 2-iP-treated plants. In shoots derived from BAP, we found upregulated genes that have already been demonstrated to be involved in de novo shoot proliferation development in several plant species; CK receptors (AHK3, ARR1), stem cell maintenance (STM, REV and CLV3), cell cycle regulation (CDKA-CYCD3 complex), as well as the auxin response factor (ARF5) and CK metabolism (CKX1). In contrast, in the 2-iP culture medium, there was an upregulation of genes involved in shoot repression (BRC1, MAX3), ARR4, a type A-response regulator (RR), and auxin metabolism (SHY2).

Exploring the Potential Role of Ribosomal Proteins to Enhance Potato Resilience in the Face of Changing Climatic Conditions.

Potatoes have emerged as a key non-grain crop for food security worldwide. However, the looming threat of climate change poses significant risks to this vital food source, particularly through the projected reduction in crop yields under warmer temperatures. To mitigate potential crises, the development of potato varieties through genome editing holds great promise. In this study, we performed a comprehensive transcriptomic analysis to investigate microtuber development and identified several differentially expressed genes, with a particular focus on ribosomal proteins-RPL11, RPL29, RPL40 and RPL17. Our results reveal, by protein-protein interaction (PPI) network analyses, performed with the highest confidence in the STRING database platform (v11.5), the critical involvement of these ribosomal proteins in microtuber development, and highlighted their interaction with PEBP family members as potential microtuber activators. The elucidation of the molecular biological mechanisms governing ribosomal proteins will help improve the resilience of potato crops in the face of today's changing climatic conditions.

CRISPRa-mediated transcriptional activation of the SlPR-1 gene in edited tomato plants.

With the continuous deterioration of arable land due to an ever-growing population, improvement of crops and crop protection have a fundamental role in maintaining and increasing crop productivity. Alternatives to the use of pesticides encompass the use of biological control agents, generation of new resistant crop cultivars, the application of plant activator agrochemicals to enhance plant defenses, and the use of gene editing techniques, like the CRISPR-Cas system. Here, we test the hypothesis that epigenome editing, via CRISPR activation (CRISPRa), activate tomato plant defense genes to confer resistance against pathogen attack. We provide evidence that edited tomato plants for the PATHOGENESIS-RELATED GENE 1 gene (SlPR-1) show enhanced disease resistance to Clavibacter michiganensis subsp. michiganensis infection. Resistance was assessed by evaluating disease progression and symptom appearance, pathogen accumulation, and changes in SlPR-1 gene expression at different time points. We determined that CRISPRa-edited plants develop enhanced disease-resistant to the pathogen without altering their agronomic characteristics and, above all, preventing the advancement of disease symptoms, stem canker, and plant death.

Solanum tuberosum Microtuber Development under Darkness Unveiled through RNAseq Transcriptomic Analysis.

Potato microtuber (MT) development through in vitro techniques are ideal propagules for producing high quality potato plants. MT formation is influenced by several factors, i.e., photoperiod, sucrose, hormones, and osmotic stress. We have previously developed a protocol of MT induction in medium with sucrose (8% w/v), gelrite (6g/L), and 2iP as cytokinin under darkness. To understand the molecular mechanisms involved, we performed a transcriptome-wide analysis. Here we show that 1715 up- and 1624 down-regulated genes were involved in this biological process. Through the protein-protein interaction (PPI) network analyses performed in the STRING database (v11.5), we found 299 genes tightly associated in 14 clusters. Two major clusters of up-regulated proteins fundamental for life growth and development were found: 29 ribosomal proteins (RPs) interacting with 6 PEBP family members and 117 cell cycle (CC) proteins. The PPI network of up-regulated transcription factors (TFs) revealed that at least six TFs-MYB43, TSF, bZIP27, bZIP43, HAT4 and WOX9-may be involved during MTs development. The PPI network of down-regulated genes revealed a cluster of 83 proteins involved in light and photosynthesis, 110 in response to hormone, 74 in hormone mediate signaling pathway and 22 related to aging.

Tec1, a member of the TEA transcription factors family, is involved in virulence and basidiocarp development in Ustilago maydis.

The life cycle of Ustilago maydis involves alternation of a haploid saprophytic yeast-like stage and a dikaryotic hyphal virulent form. Under in vitro conditions, basidiocarps are formed. Analysis of the transcriptional network of basidiocarp formation revealed the possible involvement of a Tec transcription factor (Tec1, UMAG_02835) in the process. In some Ascomycota, Tec factors are involved in mycelial formation, pathogenesis, and interaction with other regulatory elements, but their role in Basidiomycota species is almost unknown. Accordingly, we proceeded to determine the role of this gene in U. maydis by its mutation. Tec1 was found to be a crucial factor for normal mating, basidiocarp development, and virulence, all of the functions related to the dikaryotic stage dependent of the b genes, whereas dimorphism and resistance to different stress conditions occurring in the haploid stage were not affected in tec1 mutants. The observation that mutants showed a low residual wild-type phenotype suggests the presence of a secondary mechanism that partially compensates the loss of Tec1.

Coffea arabica L. Resistant to Coffee Berry Borer (Hypothenemus hampei) Mediated by Expression of the Bacillus thuringiensis Cry10Aa Protein.

Coffea spp. are tropical plants used for brewing beverages from roasted and grounded seeds, the favorite drink in the world. It is the most important commercial crop plant and the second most valuable international commodity after oil. Global coffee trade relies on two Coffea species: C. arabica L. (arabica coffee) comprising 60% and C. canephora (robusta) comprising the remaining 40%. Arabica coffee has lower productivity and better market price than robusta. Arabica coffee is threatened by disease (i.e., coffee leaf rust), pests [i.e., Hypothenemus hampei or coffee berry borer (CBB) and nematodes], and susceptibility to climate change (i.e., drought and aluminum toxicity). Plant biotechnology by means of tissue culture inducing somatic embryogenesis (SE) process, genetic transformation, and genome editing are tools that can help to solve, at least partially, these problems. This work is the continuation of a protocol developed for stable genetic transformation and successful plant regeneration of arabica coffee trees expressing the Bacillus thuringiensis (Bt) toxin Cry10Aa to induce CBB resistance. A highly SE line with a high rate of cell division and conversion to plants with 8-month plant regeneration period was produced. To validate this capability, gene expression analysis of master regulators of SE, such as BABY BOOM (BBM), FUS3, and LEC1, embryo development, such as EMB2757, and cell cycle progression, such as ETG1 and MCM4, were analyzed during induction and propagation of non-competent and highly competent embryogenic lines. The particle bombardment technique was used to generate stable transgenic lines after 3 months under selection using hygromycin as selectable marker, and 1 month in plant regeneration. Transgenic trees developed fruits after 2 years and demonstrated expression of the Bt toxin ranging from 3.25 to 13.88 µg/g fresh tissue. Bioassays with transgenic fruits on CBB first instar larvae and adults induced mortalities between 85 and 100% after 10 days. In addition, transgenic fruits showed a seed damage lower than 9% compared to 100% of control fruits and adult mortality. This is the first report on stable transformation and expression of the Cry10Aa protein in coffee plants with the potential to control CBB.

Gene Expression Analysis of Microtubers of Potato Solanum tuberosum L. Induced in Cytokinin Containing Medium and Osmotic Stress.

Potato microtuber productions through in vitro techniques are ideal propagules for producing high quality seed potatoes. Microtuber development is influenced by several factors, i.e., high content sucrose and cytokinins are among them. To understand a molecular mechanism of microtuberization using osmotic stress and cytokinin signaling will help us to elucidate this process. We demonstrate in this work a rapid and efficient protocol for microtuber development and gene expression analysis. Medium with high content of sucrose and gelrite supplemented with 2iP as cytokinin under darkness condition produced the higher quantity and quality of microtubers. Gene expression analysis of genes involved in the two-component signaling system (StHK1), cytokinin signaling, (StHK3, StHP4, StRR1) homeodomains (WUSCHEL, POTH1, BEL5), auxin signaling, ARF5, carbon metabolism (TPI, TIM), protein synthesis, NAC5 and a morphogenetic regulator of tuberization (POTH15) was performed by qPCR real time. Differential gene expression was observed during microtuber development. Gene regulation of two component and cytokinin signaling is taking place during this developmental process, yielding more microtubers. Further analysis of each component is required to elucidate it.

Proteomic analysis and interactions network in leaves of mycorrhizal and nonmycorrhizal sorghum plants under water deficit.

For understanding the water deficit stress mechanism in sorghum, we conducted a physiological and proteomic analysis in the leaves of Sorghum bicolor L. Moench (a drought tolerant crop model) of non-colonized and colonized plants with a consortium of arbuscular mycorrhizal fungi. Physiological results indicate that mycorrhizal fungi association enhances growth and photosynthesis in plants, under normal and water deficit conditions. 2D-electrophoresis profiles revealed 51 differentially accumulated proteins in response to water deficit, of which HPLC/MS successfully identified 49. Bioinformatics analysis of protein-protein interactions revealed the participation of different metabolic pathways in nonmycorrhizal compared to mycorrhizal sorghum plants under water deficit. In noninoculated plants, the altered proteins are related to protein synthesis and folding (50S ribosomal protein L1, 30S ribosomal protein S10, Nascent polypeptide-associated complex subunit alpha), coupled with multiple signal transduction pathways, guanine nucleotide-binding beta subunit (Rack1) and peptidyl-prolyl-cis-trans isomerase (ROC4). In contrast, in mycorrhizal plants, proteins related to energy metabolism (ATP synthase-24kDa, ATP synthase ß), carbon metabolism (malate dehydrogenase, triosephosphate isomerase, sucrose-phosphatase), oxidative phosphorylation (mitochondrial-processing peptidase) and sulfur metabolism (thiosulfate/3-mercaptopyruvate sulfurtransferase) were found. Our results provide a set of proteins of different metabolic pathways involved in water deficit produced by sorghum plants alone or associated with a consortium of arbuscular mycorrhizal fungi isolated from the tropical rain forest Los Tuxtlas Veracruz, México.

Analysis of the photoreceptors involved in the light-depending basidiocarp formation in Ustilago maydis.

We have described that formation of basidiocarps by Ustilago maydis requires illumination. In the current research, we have proceeded to analyze what kind of light receptors are involved in this phenomenon. Accordingly, we investigated whether the homologues of the White Collar (WC), and the phytochrome (PHY) genes played a role in this process. Mutants deficient in either one of the three U. maydis WC homologue genes (WCO1a, WCO1b, WCO2), or the phytochrome-encoding the PHY gene were obtained. Phenotypic analysis of the mutants showed that ∆wco1a mutants formed similar numbers of basidiocarps than wild-type strain, whereas ∆wco1b mutants were severely affected in basidiocarp formation when illuminated with white, blue or red light. ∆wco2 and ∆phy1 mutants did not form basidiocarps under any illumination condition. These data indicate that Wco1a is the main blue light receptor, and Wco1b may operate as a secondary blue light receptor; Phy1 is the red light receptor, and Wco2 the transcription factor that controls the photo stimulation of the genes involved in the formation of fruiting bodies. It is suggested that effectiveness of the light receptors depends on the whole structure of the complex, possibly, because their association is necessary to maintain their functional structure.

Somatic Embryogenesis in Common BeanPhaseolus vulgaris L.

Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to induce somatic embryogenesis (SE) under in vitro conditions. An alternative strategy to yield SE is based upon the use of a cytokinin (benzyladenine) coupled with osmotic stress adaptation instead of the auxin-inducing SE in common bean. Here we described the induction of proembryogenic masses (PEM) derived from the apical meristem and cotyledonary zone of zygotic embryos, from which secondary SE indirect embryogenesis emerged. Maturation of SE was achieved by using osmotic stress medium and converted to plants. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and biobalistics as well as basic biochemical and molecular biology research.

Stress induced acquisition of somatic embryogenesis in common bean Phaseolus vulgaris L.

Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to induce somatic embryogenesis (SE) under in vitro conditions. We used an alternative strategy to induce SE in common bean based upon the use of a cytokinin (BAP) coupled with osmotic stress adaptation instead of SE response that is induced by auxins. Explants derived from zygotic embryos of common bean were subjected to osmotic stress (sucrose 12 % w/v, 0.5 M) in the presence of BAP 10 mg/L and adenine free base 40 mg/L to induce somatic embryos from specific competent cells of the apical meristem and cotyledonary node. Somatic embryos were obtained from the competent cells in a direct response (direct SE). In a secondary response (secondary SE), those somatic embryos formed proembryogenic masses (PEM) that originated/developed into secondary somatic embryos and showed the SE ontogeny. Maturation of somatic embryos was achieved by using different osmolality media and converted to plants. Full-visible light spectrum was necessary to achieve efficient plant regeneration. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and bioballistics as well as for basic biochemical and molecular biology experiments.

Characterization of the pumpkin Translationally-Controlled Tumor Protein CmTCTP.

In higher plants, the phloem plays a central role in the delivery of nutrients and signals from source to sink tissues. These signals likely coordinate different aspects of plant development, as well as its response to environmental cues. Although some phloem-transported proteins and RNAs may function as signaling molecules in plants, their mode of action remains poorly understood. Previous analysis of transcripts from CMV-infected pumpkin (Cucurbita maxima cv Big Max) identified a Translationally-Controlled Tumor Protein (TCTP) mRNA homolog, designated CmTCTP. In the present work this transcript was analyzed in terms of its expression pattern. This RNA accumulates, both in healthy and CMV-infected plants, in developing and mature phloem in petiole and roots, as well as in apices at high levels. The protein was present at lower levels in most cell types, and almost no signal was detected in apices, suggesting translational regulation of this RNA. Additionally, CmTCTP harbored by Agrobacterium rhizogenes is capable of inducing whole plant regeneration. These data suggest a role for CmTCTP in growth regulation, possibly through long-distance signaling.

The Arabidopsis thaliana SET-domain-containing protein ASHH1/SDG26 interacts with itself and with distinct histone lysine methyltransferases.

Polycomb group (PcG) and trithorax group (trxG) proteins are key regulators of homeotic genes and have central roles in cell proliferation, growth and development. In animals, PcG and trxG proteins form higher order protein complexes that contain SET domain proteins with histone methyltransferase activity, and are responsible for the different types of lysine methylation at the N-terminal tails of the core histone proteins. However, whether H3K4 methyltransferase complexes exist in Arabidopsis thaliana remains unknown. Here, we make use of the yeast two-hybrid system and the bimolecular fluorescence complementation assay to provide evidence for the self-association of the Arabidopsis thaliana SET-domain-containing protein SET DOMAIN GROUP 26 (SDG26), also known as ABSENT, SMALL, OR HOMEOTIC DISCS 1 HOMOLOG 1 (ASHH1). In addition, we show that the ASHH1 protein associates with SET-domain-containing sequences from two distinct histone lysine methyltransferases, the ARABIDOPSIS HOMOLOG OF TRITHORAX-1 (ATX1) and ASHH2 proteins. Furthermore, after screening a cDNA library we found that ASHH1 interacts with two proteins from the heat shock protein 40 kDa (Hsp40/DnaJ) superfamily, thus connecting the epigenetic network with a system sensing external cues. Our findings suggest that trxG complexes in Arabidopsis thaliana could involve different sets of histone lysine methyltransferases, and that these complexes may be engaged in multiple developmental processes in Arabidopsis.

Ripening in papaya fruit is altered by ACC oxidase cosuppression.

Papaya (Carica papaya) is a very important crop in many tropical countries but it is highly susceptible to parasitic diseases, physiological disorders, mechanical damage and fruit overripening. Here we report a study on ACC oxidase cosuppression and its effects on papaya fruit ripening. Papaya ACC oxidase was isolated using PCR and embriogenic cells transformed by biolistic using the CaMV 35S promoter to drive the expression of the PCR fragment in sense orientation. Fifty transgenic lines were recovered and 20 of those were grown under field conditions. Southern analysis showed incorporation of the transgene in different copy numbers in the papaya genome. Fruits were evaluated in terms of texture (firmness), colour development, respiration and ethylene production. A sharp reduction in ethylene and CO2 production was detected, whereas softening and colour development of the peel were also altered. Overall, transgenic fruits showed a delay in ripening rate. A reduction in mRNA level for ACC oxidase in transgenic fruit was clearly detectable by northern blot. More studies are necessary before this technology can be used to extend the shelf life of papaya fruit.

A new highly effective anticysticercosis vaccine expressed in transgenic papaya.

The use of transgenic plants as new antigen-delivery systems for subunit vaccines has been increasingly explored. We herein report progress toward a papaya-based vaccine against cysticercosis. Synthetic peptides (KETc1, KETc12, KETc7) were successfully expressed in 19 different transgenic papaya clones and found to be immunogenic. Complete protection against cysticercosis was induced with the soluble extract of the clones that expressed the higher levels of transcripts in up to 90% of the immunized mice. This study represents a key step towards the development of a more effective, sustainable and affordable oral subunit vaccine against human and pig cysticercosis.

Improvement of the synthetic tri-peptide vaccine (S3Pvac) against porcine Taenia solium cysticercosis in search of a more effective, inexpensive and manageable vaccine.

Vaccination of pigs may curtail Taenia solium transmission by reducing the number of cysticerci, the precursors of adult intestinal tapeworms in humans. Several antigen preparations induce protection against porcine cysticercosis in experimental settings but only one subunit vaccine (S3Pvac) has been tested and proved effective in the field against naturally acquired disease. Besides improving of the vaccine's effectiveness, significant reductions in production costs and in the logistics of its administration are necessary for the feasibility of nationwide control programs. This review highlights the development of several versions of S3Pvac aimed to increase effectiveness, reduce costs and increase feasibility by novel delivery systems and alternative routes of administration.

An alternative pathway for plant in vitro regeneration of chinaberry -tree Melia azedarach L. derived from the induction of somatic embryogenesis

A highly efficient somatic embryogenesis system and subsequent plant regeneration of chinaberry (Melia azedarach L.) was developed. Plants were regenerated from indirect somatic embryogenesis induction. Novel features of this improved protocol, include: a) Embryogenic callus induction with no addition of 2, 4-D in the culture media; b) Somatic embryos differentiation was achieved by using high concentration of cytokinins (BAP 10 mg/L) and adenine; c) 100 percent conversion of somatic embryos to plants was practically obtained and 100 percent of plants survived under greenhouse conditions; d) Addition of putrescine improved somatic embryos germination. The amount of somatic embryos produced by the pathway of indirect somatic embryogenesis was 447 per gram of fresh weight callus. Regenerated plants were phenotypically normal. The developed protocol established the potential to produce plantlets from cotyledon explants through somatic embryogenesis. It also presents itself as a highly efficient method for mass clonal propagation and conservation of Melia azedarach.

Stable genetic transformation of the ectomycorrhizal fungus Pisolithus tinctorius.

In the present work the genetic transformation and the expression of gene markers in transgenic Pisolithus tinctorius are reported. The ectomycorrhizae are facultative symbionts of plant roots, which are capable of affording mineral nutrients to its co-host in exchange of fixed carbon. Given the importance of this association (more than 80% of gymnosperms are associated with these fungi), its study from both basic and applied viewpoints is relevant. We have transformed this fungus with reporter genes and analyzed their expression in its saprophytic state. Genetic transformation was performed by microprojectile bombardment and Agrobacterium-mediated transformation. This last method proved to be the more efficient. Southern analysis of biolistic-transformed fungi revealed the random integration of the transgene into the genome. The accumulation of the transcript of the reporter gene was demonstrated by RT-PCR. The visualization of GFP-associated fluorescence in saprophytic mycelia confirmed the expression of the reporter gene. This is the first report on the stable transformation and expression of GFP in the ectomycorrhizal fungus P. tinctorius.

Infection of alternative host plant species by Ustilago maydis.

• Here, the host specificity of the corn smut fungus Ustilago maydis was analyzed, with the long-term objective of understanding the different aspects of its pathogenic behavior. • Axenic plantlets obtained in vitro, including one gymnosperm, monocotyledons and dicotyledons, were inoculated with a diploid strain of U. maydis, incubated in a growth chamber, and observed periodically. • All plants were susceptible to infection. The most common symptoms were growth of fungal mycelium on stems and leaves, increase in root number in monocots, or development of adventitious roots in dicots. Other symptoms - chlorosis, increased anthocyanins, necrosis and stunting - varied among the different plant species. Ustilago penetrated and grew into the plant tissues in the form of pleomorphic mycelium, but no teliospores were formed. Noticeably, the fungus induced formation of lateral buds and tumors in papaya. • The results provide evidence that U. maydis is able to infect a variety of phylogenetically unrelated plants grown under axenic conditions. These results may be useful in the analysis of different phenomena associated with the complex pathogenic behavior of U. maydis.