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Protoplasma May 2024Phytophthora cinnamomi is an oomycete plant pathogen with a host range of almost 5000 plant species worldwide and therefore poses a serious threat to biodiversity. Omics...
Phytophthora cinnamomi is an oomycete plant pathogen with a host range of almost 5000 plant species worldwide and therefore poses a serious threat to biodiversity. Omics technology has provided significant progress in our understanding of oomycete biology, however, transformation studies of Phytophthora for gene functionalisation are still in their infancy. Only a limited number of Phytophthora species have been successfully transformed and gene edited to elucidate the role of particular genes. There is a need to escalate our efforts to understand molecular processes, gene regulation and infection mechanisms of the pathogen to enable us to develop new disease management strategies. The primary obstacle hindering the advancement of transformation studies in Phytophthora is their challenging and unique nature, coupled with our limited comprehension of why they remain such an intractable system to work with. In this study, we have identified some of the key factors associated with the recalcitrant nature of P. cinnamomi. We have incorporated fluorescence microscopy and flow cytometry along with the organelle-specific dyes, fluorescein diacetate, Hoechst 33342 and MitoTracker™ Red CMXRos, to assess P. cinnamomi-derived protoplast populations. This approach has also provided valuable insights into the broader cell biology of Phytophthora. Furthermore, we have optimized the crucial steps that allow transformation of P. cinnamomi and have generated transformed isolates that express a cyan fluorescent protein, with a transformation efficiency of 19.5%. We therefore provide a platform for these methodologies to be applied for the transformation of other Phytophthora species and pave the way for future gene functionalisation studies.
PubMed: 38702562
DOI: 10.1007/s00709-024-01953-y -
BMC Plant Biology Apr 2024The La-related proteins (LARPs) are a superfamily of RNA-binding proteins associated with regulation of gene expression. Evidence points to an important role for...
BACKGROUND
The La-related proteins (LARPs) are a superfamily of RNA-binding proteins associated with regulation of gene expression. Evidence points to an important role for post-transcriptional control of gene expression in germinating pollen tubes, which could be aided by RNA-binding proteins.
RESULTS
In this study, a genome-wide investigation of the LARP proteins in eight plant species was performed. The LARP proteins were classified into three families based on a phylogenetic analysis. The gene structure, conserved motifs, cis-acting elements in the promoter, and gene expression profiles were investigated to provide a comprehensive overview of the evolutionary history and potential functions of ZmLARP genes in maize. Moreover, ZmLARP6c1 was specifically expressed in pollen and ZmLARP6c1 was localized to the nucleus and cytoplasm in maize protoplasts. Overexpression of ZmLARP6c1 enhanced the percentage pollen germination compared with that of wild-type pollen. In addition, transcriptome profiling analysis revealed that differentially expressed genes included PABP homologous genes and genes involved in jasmonic acid and abscisic acid biosynthesis, metabolism, signaling pathways and response in a Zmlarp6c1::Ds mutant and ZmLARP6c1-overexpression line compared with the corresponding wild type.
CONCLUSIONS
The findings provide a basis for further evolutionary and functional analyses, and provide insight into the critical regulatory function of ZmLARP6c1 in maize pollen germination.
Topics: Zea mays; Plant Proteins; Pollen; Gene Expression Profiling; Phylogeny; Gene Expression Regulation, Plant; Multigene Family; Genome, Plant; RNA-Binding Proteins
PubMed: 38684961
DOI: 10.1186/s12870-024-05054-z -
Plants (Basel, Switzerland) Apr 2024In rice, there is a lack of comprehensive research on the functional aspects of the members of the () gene family. This study provides a comprehensive investigation of...
In rice, there is a lack of comprehensive research on the functional aspects of the members of the () gene family. This study provides a comprehensive investigation of the gene family, covering phylogeny, gene structure, promoter analysis, expression analysis, subcellular localization, and protein interaction. Remarkably, we discovered a specific gene loss event occurred in the chloroplast-localized group IIa SHMTs in monocotyledons. However, OsSHMT3, which originally classified within cytoplasmic-localized group Ib, was found to be situated within chloroplasts in rice protoplasts. All five OsSHMTs are capable of forming homodimers, with OsSHMT3 being the only one able to form dimers with other OsSHMTs, except for OsSHMT1. It is proposed that OsSHMT3 functions as a mobile protein, collaborating with other OsSHMT proteins. Furthermore, the results of -acting element prediction and expression analysis suggested that members of the family could be involved in diverse stress responses and hormone regulation. Our study aims to provide novel insights for the future exploration of SHMTs.
PubMed: 38674525
DOI: 10.3390/plants13081116 -
Plant Methods Apr 2024
PubMed: 38659047
DOI: 10.1186/s13007-024-01176-5 -
Methods in Molecular Biology (Clifton,... 2024Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to...
Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.
Topics: Arabidopsis; Protoplasts; Plant Leaves; Protein Interaction Mapping; Arabidopsis Proteins; Microscopy, Fluorescence; Luminescent Proteins; Nicotiana; Protein Binding; Agrobacterium
PubMed: 38656499
DOI: 10.1007/978-1-0716-3778-4_21 -
Plant Physiology Apr 2024The bundle sheath cell (BSC) layer tightly enveloping the xylem throughout the leaf is recognized as a major signal-perceiving "valve" in series with stomata, regulating...
The bundle sheath cell (BSC) layer tightly enveloping the xylem throughout the leaf is recognized as a major signal-perceiving "valve" in series with stomata, regulating leaf hydraulic conductance (Kleaf) and thereby radial water flow via the transpiring leaf. The BSC blue light (BL) signaling pathway increases Kleaf and the underlying BSC water permeability. Here, we explored the hypothesis that BSCs also harbor a Kleaf-downregulating signaling pathway related to the stress phytohormone abscisic acid (ABA). We employed fluorescence imaging of xylem sap in detached leaves and BSC protoplasts from different genotypes of Arabidopsis (Arabidopsis thaliana) plants, using pH and membrane potential probes to monitor physiological responses to ABA and BL in combination with pharmacological agents. We found that BL-enhanced Kleaf required elevated BSC cytosolic Ca2+. ABA inhibited BL-activated xylem-sap-acidifying BSC H + -ATPase AHA2 (Arabidopsis H + -ATPase 2), resulting in depolarized BSCs and alkalinized xylem sap. ABA also stimulated BSC vacuolar H + -ATPase (VHA), which alkalinized the BSC cytosol. Each pump stimulation, AHA2 by BL and VHA by ABA (under BL), also required Ca2+. ABA stimulated VHA in the dark depending on Ca2+, but only in an alkaline external medium. Taken together with earlier findings on the pH sensitivity of BSC osmotic water permeability (i.e., aquaporin activity), our results suggest a Ca2+-dependent and pH-mediated causative link between the BL- and ABA-regulated activities of two BSC H + -ATPases and Kleaf.
PubMed: 38652805
DOI: 10.1093/plphys/kiae226 -
World Journal of Microbiology &... Apr 2024The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides...
The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.
Topics: Gene Editing; CRISPR-Cas Systems; Ascomycota; Endophytes; Melanins; Fungal Proteins; Protoplasts
PubMed: 38652405
DOI: 10.1007/s11274-024-03988-y -
The Plant Journal : For Cell and... Jul 2024The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several...
The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotype used to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.
Topics: Vitis; Gene Editing; CRISPR-Cas Systems; Protoplasts; Genotype; Lipids; Ribonucleoproteins; Wine; Genome, Plant; Oxidoreductases
PubMed: 38646817
DOI: 10.1111/tpj.16770 -
The Plant Journal : For Cell and... Apr 2024Green feather algae (Bryopsidales) undergo a unique life cycle in which a single cell repeatedly executes nuclear division without cytokinesis, resulting in the...
Green feather algae (Bryopsidales) undergo a unique life cycle in which a single cell repeatedly executes nuclear division without cytokinesis, resulting in the development of a thallus (>100 mm) with characteristic morphology called coenocyte. Bryopsis is a representative coenocytic alga that has exceptionally high regeneration ability: extruded cytoplasm aggregates rapidly in seawater, leading to the formation of protoplasts. However, the genetic basis of the unique cell biology of Bryopsis remains poorly understood. Here, we present a high-quality assembly and annotation of the nuclear genome of Bryopsis sp. (90.7 Mbp, 27 contigs, N50 = 6.7 Mbp, 14 034 protein-coding genes). Comparative genomic analyses indicate that the genes encoding BPL-1/Bryohealin, the aggregation-promoting lectin, are heavily duplicated in Bryopsis, whereas homologous genes are absent in other ulvophyceans, suggesting the basis of regeneration capability of Bryopsis. Bryopsis sp. possesses >30 kinesins but only a single myosin, which differs from other green algae that have multiple types of myosin genes. Consistent with this biased motor toolkit, we observed that the bidirectional motility of chloroplasts in the cytoplasm was dependent on microtubules but not actin in Bryopsis sp. Most genes required for cytokinesis in plants are present in Bryopsis, including those in the SNARE or kinesin superfamily. Nevertheless, a kinesin crucial for cytokinesis initiation in plants (NACK/Kinesin-7II) is hardly expressed in the coenocytic part of the thallus, possibly underlying the lack of cytokinesis in this portion. The present genome sequence lays the foundation for experimental biology in coenocytic macroalgae.
PubMed: 38642374
DOI: 10.1111/tpj.16764 -
Plants (Basel, Switzerland) Apr 2024is a wild diploid tuber-bearing plant. We here demonstrate transgene-free genome editing of protoplasts and regeneration of gene-edited plants. We use...
is a wild diploid tuber-bearing plant. We here demonstrate transgene-free genome editing of protoplasts and regeneration of gene-edited plants. We use ribonucleoproteins, consisting of Cas9 and sgRNA, assembled in vitro, to target a gene belonging to the nitrate and peptide transporter family. Four different sgRNAs were designed and we observed efficiency in gene-editing in the protoplast pool between 8.5% and 12.4%. Twenty-one plants were re-generated from microcalli developed from individual protoplasts. In three of the plants we found that the target gene had been edited. Two of the edited plants had deletion mutations introduced into both alleles, whereas one only had a mutation in one of the alleles. Our work demonstrates that protocols for the transformation of can be optimized to be applied to a wild species.
PubMed: 38611572
DOI: 10.3390/plants13071044