Biology subjects

Frommer, W. B.

Publications and source records attributed to Frommer, W. B..

32 records · Page 2Linked to original sources

bioRxiv2023

Rice Yellow Mottle Virus resistance by genome editing of the Oryza sativa L. ssp. japonica nucleoporin gene OsCPR5.1 but not OsCPR5.2

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (O. glaberrima), however, introgression into O. sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single knockout mutants show neither substantial growth defects nor symptoms indicative of programmed cell death, possibly reflecting functional redundancy of the isoforms regarding other important functions. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

plant biology↗

bioRxiv2022

Genome editing of an African elite rice variety confers resistance against endemic and emerging Xanthomonas oryzae pv. oryzae strains

Bacterial leaf blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), threatens global food security and the livelihood of small-scale rice producers. Analyses of Xoo collections from Asia, Africa and the Americas demonstrated surprising continental segregation, despite robust global rice trade. Here, we report unprecedented BB outbreaks in Tanzania. The causative strains, unlike endemic Xoo, carry Asian-type TAL effectors targeting the sucrose transporter SWEET11a and suppressing Xa1. Phylogenomics clustered these strains with Xoo strains from China. African rice varieties do not carry suitable resistance genes. To protect African rice production against this emerging threat, we developed a hybrid CRISPR-Cas9/Cpf1 system to edit six TALe-binding elements in three SWEET promoters of the East African elite variety Komboka. The edited lines show broad-spectrum resistance against Asian and African strains of Xoo, including strains recently discovered in Tanzania. This strategy could help to protect global rice crops from BB pandemics.

plant biology↗

bioRxiv2022

A high confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution

Cells of multicellular organisms exchange nutrients, building blocks and information. In animals, this happens via gap junctions, in plants via plasmodesmata (PD). PD have striking properties, translocating a large range of molecules from ions, to metabolites, RNA and proteins up to 40 kDa. PD are hard to characterize due to being deeply embedded into cell walls and the presence of several membranes. While previous studies of protein composition of PD from angiosperms identified large lists of proteins, few were validated. Here, we developed a PD scoring approach in conjunction with systematic localization on a large scale to define a high-confidence PD proteome of Physcomitrium patens. This high confidence PD proteome comprises nearly 300 proteins, which together with the bona fide PD proteins from literature, are made available in the public PDDB database. Conservation of localization across plant species strengthens the reliability of plant PD proteomes and provides a basis for exploring the evolution of this important organelle. In particular, the P. patens PD proteome was highly enriched in cell wall modifying proteins. Callose-degrading glycolyl hydrolase family 17 (GHL17) proteins are presented as an abundant PD protein family with representatives across an evolutionary scale. Exclusively members of the alpha-clade of the GHL17 family are shown to be PD localized and their orthologs occur only in plant species which have developed PD. Members of the EXORDIUM-family and xyloglucan transglycosylases are additional cell-wall located proteins highly abundant in the P. patens PD proteome also showing evolutionary diversification of PD localized family members from other clade members.

plant biology↗

bioRxiv2022

SWEET13 transport of sucrose, but not gibberellin, restores male fertility in Arabidopsis sweet13;14

SWEET sucrose transporters play important roles in the allocation of sucrose in plants. Some SWEETs were shown to also mediate transport of the plant growth regulator gibberellin (GA). The close physiological relationship between sucrose and GA raised the questions of if there is a functional connection, and whether one or both of the substrates are physiologically relevant. To dissect these two activities, molecular dynamics were used to map the binding sites of sucrose and GA in the pore of SWEET13 and predicted binding interactions that might be selective for sucrose or GA. Transport assays confirmed these predictions. In transport assays, the N76Q mutant had 7x higher relative GA3 activity, and the S142N mutant only transported sucrose. The impaired pollen viability and germination in sweet13;14 double mutants were complemented by the sucrose-selective SWEET13S142N but not by the SWEET13N76Q mutant, indicating that sucrose is the physiologically relevant substrate and that GA transport capacity is dispensable in the context of male fertility. Therefore, GA supplementation to counter male sterility may act indirectly via stimulating sucrose supply in male sterile mutants. These findings are also relevant in the context of the role of SWEETs in pathogen susceptibility.

plant biology↗

bioRxiv2022

OzTracs: Optical Osmolality Reporters Engineered from Mechanosensitive Ion Channels

Interactions between physical forces and membrane proteins underpin many forms of environmental sensation and acclimation. Microbes survive sudden osmotic stresses with the help of mechanically gated ion channels and osmolyte transporters. Plant mechanosensitive ion channels have been shown to function in defense signaling. Here, we engineered genetically encoded osmolality sensors (OzTracs) by fusing green fluorescent protein spectral variants to the mechanosensitive ion channels MscL from E. coli or MSL10 from A. thaliana. When expressed in yeast cells, OzTrac sensors reported osmolality changes as a proportional change in emission ratio of the two fluorescent protein domains. Live-cell imaging revealed accumulation of fluorescent sensors in internal aggregates presumably derived from the endomembrane system. Thus, OzTrac sensors serve as osmolality-dependent reporters through an indirect mechanism, such as effects on molecular crowding or fluorophore solvation.

cell biology↗

bioRxiv2021

Sucrose-dependence of sugar uptake, quorum sensing and virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae

Withdrawal StatementThe authors have withdrawn their manuscript owing to potential issues with the qRT-PCR data and issues with accessing some of the raw data from one of the authors (FAIR principle requirements). Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

plant biology↗

bioRxiv2021

OsSWEET11b, a sixth leaf blight susceptibility gene involved in sugar transport-dependent male fertility

SWEETs play important roles in intercellular sugar transport. Induction of SWEET sugar transporters by transcription activator-like effectors (TALe) of Xanthomonas ssp. is a key factor for bacterial leaf blight (BLB) infection of rice, cassava and cotton. Here, we identified the so far unknown OsSWEET11b with roles in male fertility and BLB susceptibility in rice. While single ossweet11a or b mutants were fertile, double mutants were sterile. Since clade III SWEETs can transport gibberellin (GA), a key hormone for rice spikelet fertility, sterility and BLB susceptibility might be explained by GA transport deficiencies. However, in contrast to the Arabidopsis homologs, OsSWEET11b did not mediate detectable GA transport. Fertility and susceptibility must therefore depend on SWEET11b-mediated sucrose transport. Ectopic induction of OsSWEET11b by designer TALe enables TALe-free Xanthomonas oryzae pv. oryzae (Xoo) to cause disease, identifying OsSWEET11b as a BLB susceptibility gene and demonstrating that the induction of host sucrose uniporter activity is key to virulence of Xoo. Notably, only three of now six clade III SWEETs are targeted by known Xoo strains from Asia and Africa. The identification of OsSWEET11b has relevance in the context of fertility and for protecting rice against emerging Xoo strains that evolve TALes to exploit OsSWEET11b.

plant biology↗

bioRxiv2020

Arabidopsis bZIP11 is a susceptibility factor during Pseudomonas syringae infection

AO_SCPLOWBSTRACTC_SCPLOWThe induction of plant nutrient secretion systems is critical for successful pathogen infection. Some bacterial pathogens, e.g. Xanthomonas species, use TAL (transcription activator-like) effectors to induce transcription of SWEET sucrose efflux transporters. Pseudomonas syringae pathovar (pv.) tomato strain DC3000 lacks TAL effectors, yet is able to induce multiple SWEETs in Arabidopsis thaliana by unknown mechanisms. Since bacteria require other nutrients besides sugars for efficient reproduction, we hypothesized that Pseudomonas may depend on host transcription factors involved in secretory programs to increase access to essential nutrients. Bioinformatic analyses identified the Arabidopsis basic-leucine zipper transcription factor bZIP11 as a potential regulator of nutrient transporters, including SWEETs and UmamiT amino acid transporters. Inducible downregulation of bZIP11 expression in Arabidopsis resulted in reduced growth of P. syringae pv. tomato strain DC3000, whereas inducible overexpression of bZIP11 resulted in increased bacterial growth, supporting the hypothesis that bZIP11 regulated transcription programs are essential for maximal pathogen titer in leaves. Our data are consistent with a model in which a pathogen alters host transcription factor expression upstream of secretory transcription networks to promote nutrient efflux from host cells.

plant biology↗

bioRxiv2020

Affinity series of genetically encoded high sensitivity Förster Resonance Energy Transfer sensors for sucrose

Genetically encoded fluorescent sugar sensors are valuable tools for the discovery of transporters and for quantitative monitoring of sugar steady-state levels in intact tissues. Genetically encoded Forster Resonance Energy Transfer sensors for glucose have been designed and optimized extensively, and a full series of affinity mutants is available for in vivo studies. However, to date, only a single improved sensor FLIPsuc-90{micro}{Delta}1 with a Km for sucrose of [~]90 {micro}M is available for sucrose monitoring. This sucrose sensor was engineered on the basis of an Agrobacterium tumefaciens sugar binding protein. Here, we took a two-step approach to first systematically improve the dynamic range of the FLIPsuc nanosensor and then expand the detection range from micromolar to millimolar sucrose concentrations by mutating a key residue in the binding site. The resulting series of sucrose sensors may allow systematic investigation of sucrose transporter candidates and comprehensive in vivo analyses of sucrose concentration in plants. Since FLIPsuc-90{micro} also detects trehalose in animal cells, the new series of sensors can be used to investigate trehalose transporter candidates and monitor trehalose steady-state levels in vivo as well.

bioengineering↗

bioRxiv2020

Feedback inhibition of AMT1 NH4+-transporters mediated by CIPK15 kinase

Ammonium (NH4+), a key nitrogen form, becomes toxic when it accumulates to high levels. Ammonium transporters (AMTs) are the key transporters responsible for NH4+ uptake. AMT activity is under allosteric feedback control, mediated by phosphorylation of a threonine in the cytosolic C-terminus (CCT). However, the kinases responsible for the NH4+-triggered phosphorylation remain unknown. In this study, a functional screen identified protein kinase CBL-Interacting Protein Kinase15 (CIPK15) as a negative regulator of AMT1;1 activity. CIPK15 was able to interact with several AMT1 paralogs at the plasma membrane. Analysis of AmTryoshka, an NH4+ transporter activity sensor for AMT1;3 in yeast, and a two-electrode-voltage-clamp (TEVC) of AMT1;1 in Xenopus oocytes showed that CIPK15 inhibits AMT activity. CIPK15 transcript levels increased when seedlings were exposed to elevated NH4+ levels. Notably, cipk15 knockout mutants showed higher 15NH4+ uptake and accumulated higher amounts of NH4+ compared to the wild-type. Consistently, cipk15 was hypersensitive to both NH4+ and methylammonium but not nitrate (NO3-). Taken together, our data indicate that feedback inhibition of AMT1 activity is mediated by the protein kinase CIPK15 via phosphorylation of residues in the CCT to reduce NH4+-accumulation.

plant biology↗

bioRxiv2020

Unique and distinct identities and functions of leaf phloem cells revealed by single cell transcriptomics

AO_SCPLOWBSTRACTC_SCPLOWThe leaf vasculature plays a key role in solute translocation. Veins consist of at least seven distinct cell types, with specific roles in transport, metabolism, and signaling. Little is known about the vascular cells in leaves, in particular the phloem parenchyma (PP). PP effluxes sucrose into the apoplasm as a basis for phloem loading; yet PP has only been characterized microscopically. Here, we enriched vascular cells from Arabidopsis leaves to generate a single-cell transcriptome atlas of leaf vasculature. We identified [≥]19 cell clusters, encompassing epidermis, guard cells, hydathodes, mesophyll, and all vascular cell types, and used metabolic pathway analysis to define their roles. Clusters comprising PP cells were enriched for transporters, including SWEET11 and SWEET12 sucrose and UmamiT amino acid efflux carriers. PP development occurs independently from APL, a transcription factor required for phloem differentiation. PP cells have a unique pattern of amino acid metabolism activity distinct from companion cells (CC), explaining differential distribution/metabolism of amino acids in veins. The kinship relation of the vascular clusters is strikingly similar to the vein morphology, except for a clear separation of CC from the other vascular cells including PP. In summary, our scRNA-seq analysis provides a wide range of information into the leaf vasculature and the role and relationship of the leaf cell types.

plant biology↗

bioRxiv2020

Phloem loading via the abaxial bundle sheath cells in maize leaves

Leaves are asymmetric, with differential functionalization of abaxial and adaxial tissues. The bundle sheath (BS) surrounding the vasculature of the C3 crop barley is dorsoventrally differentiated into three domains: adaxial structural, lateral S-type, and abaxial L-type. S-type cells seem to transfer assimilates towards the phloem. Here we used single-cell RNA sequencing to investigate BS differentiation in C4 maize. Abaxial BS (abBS) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c were also identified in the phloem parenchyma (PP). Thus maize acquired a unique mechanism for phloem loading in which abBS cells provide the main pathway for apoplasmic sucrose transfer towards the phloem. This pathway predominates in veins responsible for phloem loading (rank-2 intermediate), while rank-1 intermediate and major veins export sucrose from the phloem parenchyma (PP) adjacent to the sieve element companion cell (SE/CC) complex, as in Arabidopsis. We surmise that abBS identity is subject to dorsoventral patterning and has components of PP identity. These observations provide first insights into the unique transport-specific properties of abBS cells and support for a modification to the canonical phloem loading pathway of maize, which may be generalizable to other C4 monocots.

plant biology↗

bioRxiv2020

Noxious effects of cell surface display glutamate sensors on plant growth and development

Plants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra- and intracellular glutamate concentrations in plants, we generated Arabidopsis lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high-affinity display sensor lines revealed that root meristem development, particularly the quiescent center (QC), number of lateral roots, vegetative growth and floral architecture were impacted. Notably, the severity of the phenotypes was positively correlated with the affinity of the display sensors, intimating that their ability to sequester glutamate at the surface of the plasma membrane was responsible for the defects. Root growth defects were suppressed by supplementing culture media with low levels of glutamate. Together, the data indicate that sequestration of glutamate at the cell surface either disrupts the supply of glutamate to meristematic cells and/or impairs localized glutamatergic signaling important for developmental processes. SIGNIFICANCE STATEMENTGlutamate is an important signaling molecule in animals and plants. The affinity-dependent effects of surface-displayed glutamate sensors on plant development intimate that glutamate levels in the vicinity of the plasma membrane play important roles in signaling in plants.

plant biology↗

bioRxiv2020

Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

AO_SCPLOWBSTRACTC_SCPLOWSynthetic chemical fluorescent dyes are promising tools for many applications in biology. SNAP tagging provides a unique opportunity for labeling of specific proteins in vivo with synthetic dyes for studying for example endocytosis, or super-resolution microscopy. However, despite the potential, chemical dye tagging has not been used effectively in plants. A major drawback was the limited knowledge regarding cell wall and membrane permeability of synthetic dyes. Twenty-six out of 31 synthetic dyes were taken up into BY-2 cells, eight were not taken up and can thus serve for measuring endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine (TMR) and silicon-rhodamine (SiR) 647 were used to SNAP tag -tubulin. Successful tagging was verified by live cell imaging and visualization of microtubules arrays in interphase and during mitosis. Fluorescence activation-coupled protein labeling (FAPL) with DRBG-488 was used to observe PIN2 endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the newly forming cell plate during mitosis. Together the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety to cell biological processes.

cell biology↗