MicroRNA396 negatively regulates shoot regeneration in tomato.

Numerous studies have been dedicated to genetically engineering crops to enhance their yield and quality. One of the key requirements for generating genetically modified plants is the reprogramming of cell fate. However, the efficiency of shoot regeneration during this process is highly dependent on genotypes, and the underlying molecular mechanisms remain poorly understood. Here, we identified microRNA396 (miR396) as a negative regulator of shoot regeneration in tomato. By selecting two genotypes with contrasting shoot regeneration efficiencies and analyzing their transcriptome profiles, we found that miR396 and its target transcripts, which encode GROWTH-REGULATING FACTORs (GRFs), exhibit differential abundance between high- and low-efficiency genotypes. Suppression of miR396 functions significantly improved shoot regeneration rates along with increased expression of GRFs in transformed T0 explants, suggesting that miR396 is a key molecule involved in the determination of regeneration efficiency. Notably, we also showed that co-expression of a miR396 suppressor with the gene-editing tool can be employed to generate gene-edited plants in the genotype with a low capacity for shoot regeneration. Our findings show the critical role of miR396 as a molecular barrier to shoot regeneration in tomato and suggest that regeneration efficiency can be improved by blocking this single microRNA.

Enhanced binding and inhibition of SARS-CoV-2 by a plant-derived ACE2 protein containing a fused mu tailpiece.

Infectious diseases such as Coronavirus disease 2019 (COVID-19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The angiotensin-converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS-CoV-2 infection. Inhibition of the interaction between SARS-CoV-2 and ACE2 has been proposed as a target for the prevention and treatment of COVID-19. We produced four recombinant plant-derived ACE2 isoforms with or without the mu tailpiece (µ-tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant-derived ACE2 isoforms bound whole SARS-CoV-2 virus and the isolated receptor binding domains of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of µ-tp and KDEL to the ACE2 protein (ACE2 µK) had enhanced binding activity with SARS-CoV-2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant-derived ACE2 µK protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS-CoV-2 infection. The efficient and rapid scalability of plant-derived ACE2 µK protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks.

WUSCHEL controls genotype-dependent shoot regeneration capacity in potato.

Plant cells can reprogram their fate. The combinatorial actions of auxin and cytokinin dedifferentiate somatic cells to regenerate organs, which can develop into individual plants. As transgenic plants can be generated from genetically modified somatic cells through these processes, cell fate transition is an unavoidable step in crop genetic engineering. However, regeneration capacity closely depends on the genotype, and the molecular events underlying these variances remain elusive. In the present study, we demonstrated that WUSCHEL (WUS)-a homeodomain transcription factor-determines regeneration capacity in different potato (Solanum tuberosum) genotypes. Comparative analysis of shoot regeneration efficiency and expression of genes related to cell fate transition revealed that WUS expression coincided with regeneration rate in different potato genotypes. Moreover, in a high-efficiency genotype, WUS silencing suppressed shoot regeneration. Meanwhile, in a low-efficiency genotype, regeneration could be enhanced through the supplementation of a different type of cytokinin that promoted WUS expression. Computational modeling of cytokinin receptor-ligand interactions suggested that the docking pose of cytokinins mediated by hydrogen bonding with the core residues may be pivotal for WUS expression and shoot regeneration in potatoes. Furthermore, our whole-genome sequencing analysis revealed core sequence variations in the WUS promoters that differentiate low- and high-efficiency genotypes. The present study revealed that cytokinin responses, particularly WUS expression, determine shoot regeneration efficiency in different potato genotypes.

Efficient Plant Regeneration System from Leaf Explant Cultures of Daphne genkwa via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L-1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L-1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L-1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L-1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.

Ethylene signals modulate the survival of Arabidopsis leaf explants.

BACKGROUND: Leaf explants are major materials in plant tissue cultures. Incubation of detached leaves on phytohormone-containing media, which is an important process for producing calli and regenerating plants, change their cell fate. Although hormone signaling pathways related to cell fate transition have been widely studied, other molecular and physiological events occurring in leaf explants during this process remain largely unexplored. RESULTS: Here, we identified that ethylene signals modulate expression of pathogen resistance genes and anthocyanin accumulation in leaf explants, affecting their survival during culture. Anthocyanins accumulated in leaf explants, but were not observed near the wound site. Ethylene signaling mutant analysis revealed that ethylene signals are active and block anthocyanin accumulation in the wound site. Moreover, expression of defense-related genes increased, particularly near the wound site, implying that ethylene induces defense responses possibly by blocking pathogenesis via wounding. We also found that anthocyanin accumulation in non-wounded regions is required for drought resistance in leaf explants. CONCLUSIONS: Our study revealed the key roles of ethylene in the regulation of defense gene expression and anthocyanin biosynthesis in leaf explants. Our results suggest a survival strategy of detached leaves, which can be applied to improve the longevity of explants during tissue culture.

Nucleotide-binding leucine-rich repeat network underlies nonhost resistance of pepper against the Irish potato famine pathogen Phytophthora infestans.

Nonhost resistance (NHR) is a robust plant immune response against non-adapted pathogens. A number of nucleotide-binding leucine-rich repeat (NLR) proteins that recognize non-adapted pathogens have been identified, although the underlying molecular mechanisms driving robustness of NHR are still unknown. Here, we screened 57 effectors of the potato late blight pathogen Phytophthora infestans in nonhost pepper (Capsicum annuum) to identify avirulence effector candidates. Selected effectors were tested against 436 genome-wide cloned pepper NLRs, and we identified multiple functional NLRs that recognize P. infestans effectors and confer disease resistance in the Nicotiana benthamiana as a surrogate system. The identified NLRs were homologous to known NLRs derived from wild potatoes that recognize P. infestans effectors such as Avr2, Avrblb1, Avrblb2, and Avrvnt1. The identified CaRpi-blb2 is a homologue of Rpi-blb2, recognizes Avrblb2 family effectors, exhibits feature of lineage-specifically evolved gene in microsynteny and phylogenetic analyses, and requires pepper-specific NRC (NLR required for cell death)-type helper NLR for proper function. Moreover, CaRpi-blb2-mediated hypersensitive response and blight resistance were more tolerant to suppression by the PITG_15 278 than those mediated by Rpi-blb2. Combined results indicate that pepper has stacked multiple NLRs recognizing effectors of non-adapted P. infestans, and these NLRs could be more tolerant to pathogen-mediated immune suppression than NLRs derived from the host plants. Our study suggests that NLRs derived from nonhost plants have potential as untapped resources to develop crops with durable resistance against fast-evolving pathogens by stacking the network of nonhost NLRs into susceptible host plants.

Nuclear OsFKBP20-1b maintains SR34 stability and promotes the splicing of retained introns upon ABA exposure in rice.

Alternative splicing (AS) is a critical means by which plants respond to changes in the environment, but few splicing factors contributing to AS have been reported and functionally characterized in rice (Oryza sativa L.). Here, we explored the function and molecular mechanism of the spliceosome-associated protein OsFKBP20-1b during AS. We determined the AS landscape of wild-type and osfkbp20-1b knockout plants upon abscisic acid (ABA) treatment by transcriptome deep sequencing. To capture the dynamics of translating intron-containing mRNAs, we blocked transcription with cordycepin and performed polysome profiling. We also analyzed whether OsFKBP20-1b and the splicing factors OsSR34 and OsSR45 function together in AS using protoplast transfection assays. We show that OsFKBP20-1b interacts with OsSR34 and regulates its stability, suggesting a role as a chaperone-like protein in the spliceosome. OsFKBP20-1b facilitates the splicing of mRNAs with retained introns after ABA treatment; some of these mRNAs are translatable and encode functional transcriptional regulators of stress-responsive genes. In addition, interacting proteins, OsSR34 and OsSR45, regulate the splicing of the same retained introns as OsFKBP20-1b after ABA treatment. Our findings reveal that spliceosome-associated immunophilin functions in alternative RNA splicing in rice by positively regulating the splicing of retained introns to limit ABA response.

The spliceophilin CYP18-2 is mainly involved in the splicing of retained introns under heat stress in Arabidopsis.

Peptidyl-prolyl isomerase-like 1 (PPIL1) is associated with the human spliceosome complex. However, its function in pre-mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18-2 (AtCYP18-2), a PPIL1 homolog, plays an essential role in heat tolerance by regulating pre-mRNA splicing. Under heat stress conditions, AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP18-2 interacts with several spliceosome complex BACT components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress. The AtCYP18-2 loss-of-function allele cyp18-2 engineered by CRISPR/Cas9-mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type. Moreover, global transcriptome profiling showed that the cyp18-2 mutation affects alternative splicing of heat stress-responsive genes under heat stress conditions, particularly intron retention (IR). The abundance of most intron-containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18-2 compared to the wild type. Furthermore, the intron-containing transcripts of two heat stress-related genes, HEAT SHOCK PROTEIN 101 (HSP101) and HEAT SHOCK FACTOR A2 (HSFA2), produced functional proteins. HSP101-IR-GFP localization was responsive to heat stress, and HSFA2-III-IR interacted with HSF1 and HSP90.1 in plant cells. Our findings reveal that CYP18-2 functions as a splicing factor within the BACT spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.

Submergence promotes auxin-induced callus formation through ethylene-mediated post-transcriptional control of auxin receptors.

Plant cells in damaged tissue can be reprogrammed to acquire pluripotency and induce callus formation. However, in the aboveground organs of many species, somatic cells that are distal to the wound site become less sensitive to auxin-induced callus formation, suggesting the existence of repressive regulatory mechanisms that are largely unknown. Here we reveal that submergence-induced ethylene signals promote callus formation by releasing post-transcriptional silencing of auxin receptor transcripts in non-wounded regions. We determined that short-term submergence of intact seedlings induces auxin-mediated cell dedifferentiation across the entirety of Arabidopsis thaliana explants. The constitutive triple response 1-1 (ctr1-1) mutation induced callus formation in explants without submergence, suggesting that ethylene facilitates cell dedifferentiation. We show that ETHYLENE-INSENSITIVE 2 (EIN2) post-transcriptionally regulates the abundance of transcripts for auxin receptor genes by facilitating microRNA393 degradation. Submergence-induced calli in non-wounded regions were suitable for shoot regeneration, similar to those near the wound site. We also observed submergence-promoted callus formation in Chinese cabbage (Brassica rapa), indicating that this may be a conserved mechanism in other species. Our study identifies previously unknown regulatory mechanisms by which ethylene promotes cell dedifferentiation and provides a new approach for boosting callus induction efficiency in shoot explants.

Editing of StSR4 by Cas9-RNPs confers resistance to Phytophthora infestans in potato.

Potato (Solanum tuberosum L.) cultivation is threatened by various environmental stresses, especially disease. Genome editing technologies are effective tools for generating pathogen-resistant potatoes. Here, we established an efficient RNP-mediated CRISPR/Cas9 genome editing protocol in potato to develop Phytophthora infestans resistant mutants by targeting the susceptibility gene, Signal Responsive 4 (SR4), in protoplasts. Mutations in StSR4 were efficiently introduced into the regenerated potato plants, with a maximum efficiency of 34%. High co-expression of StEDS1 and StPAD4 in stsr4 mutants induced the accumulation of salicylic acid (SA), and enhanced the expression of the pathogen resistance marker StPR1. In addition, increased SA content in the stsr4 mutant enhanced its resistance to P. infestans more than that in wild type. However, the growth of stsr4_3-19 and stsr4_3-698 mutants with significantly high SA was strongly inhibited, and a dwarf phenotype was induced. Therefore, it is important to adequate SA accumulation in order to overcome StSR4 editing-triggered growth inhibition and take full advantages of the improved pathogen resistance of stsr4 mutants. This RNP-mediated CRISPR/Cas9-based potato genome editing protocol will accelerate the development of pathogen-resistant Solanaceae crops via molecular breeding.

The Arabidopsis cyclophilin CYP18-1 facilitates PRP18 dephosphorylation and the splicing of introns retained under heat stress.

In plants, heat stress induces changes in alternative splicing, including intron retention; these events can rapidly alter proteins or downregulate protein activity, producing nonfunctional isoforms or inducing nonsense-mediated decay of messenger RNA (mRNA). Nuclear cyclophilins (CYPs) are accessory proteins in the spliceosome complexes of multicellular eukaryotes. However, whether plant CYPs are involved in pre-mRNA splicing remain unknown. Here, we found that Arabidopsis thaliana CYP18-1 is necessary for the efficient removal of introns that are retained in response to heat stress during germination. CYP18-1 interacts with Step II splicing factors (PRP18a, PRP22, and SWELLMAP1) and associates with the U2 and U5 small nuclear RNAs in response to heat stress. CYP18-1 binds to phospho-PRP18a, and increasing concentrations of CYP18-1 are associated with increasing dephosphorylation of PRP18a. Furthermore, interaction and protoplast transfection assays revealed that CYP18-1 and the PP2A-type phosphatase PP2A B'η co-regulate PRP18a dephosphorylation. RNA-seq and RT-qPCR analysis confirmed that CYP18-1 is essential for splicing introns that are retained under heat stress. Overall, we reveal the mechanism of action by which CYP18-1 activates the dephosphorylation of PRP18 and show that CYP18-1 is crucial for the efficient splicing of retained introns and rapid responses to heat stress in plants.

Temporally distinct regulatory pathways coordinate thermo-responsive storage organ formation in potato.

Tuberization is an important developmental process in potatoes, but it is highly affected by environmental conditions. Temperature is a major environmental factor affecting tuberization, with high temperatures suppressing tuber development. However, the temporal aspects of thermo-responsive tuberization remain elusive. In this study, we show that FT homolog StSP6A is suppressed by temporally distinct regulatory pathways. Experiments using StSP6A-overexpressing plants show that post-transcriptional regulation plays a major role at the early stage, while transcriptional regulation is an important late-stage factor, suppressing StSP6A at high temperatures in leaves. Overexpression of StSP6A in leaves restores tuber formation but does not recover tuber yield at the late stage, possibly because of suppressed sugar transport at high temperatures. Transcriptome analyses lead to the identification of potential regulators that may be involved in thermo-responsive tuberization at different stages. Our work shows that potato has temporally distinct molecular mechanisms that finely control tuber development at high temperatures.

Wound-induced signals regulate root organogenesis in Arabidopsis explants.

BACKGROUND: Reactive oxygen species (ROS) and calcium ions (Ca2+) are representative signals of plant wound responses. Wounding triggers cell fate transition in detached plant tissues and induces de novo root organogenesis. While the hormonal regulation of root organogenesis has been widely studied, the role of early wound signals including ROS and Ca2+ remains largely unknown. RESULTS: We identified that ROS and Ca2+ are required for de novo root organogenesis, but have different functions in Arabidopsis explants. The inhibition of the ROS and Ca2+ signals delayed root development in detached leaves. Examination of the auxin signaling pathways indicated that ROS and Ca2+ did not affect auxin biosynthesis and transport in explants. Additionally, the expression of key genes related to auxin signals during root organogenesis was not significantly affected by the inhibition of ROS and Ca2+ signals. The addition of auxin partially restored the suppression of root development by the ROS inhibitor; however, auxin supplementation did not affect root organogenesis in Ca2+-depleted explants. CONCLUSIONS: Our results indicate that, while both ROS and Ca2+ are key molecules, at least in part of the auxin signals acts downstream of ROS signaling, and Ca2+ acts downstream of auxin during de novo root organogenesis in leaf explants.

Construction of SARS-CoV-2 virus-like particles in plant.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

Corrigendum: Editing of StSR4 by Cas9-RNPs confers resistance to Phytophthora infestans in potato.

[This corrects the article DOI: 10.3389/fpls.2022.997888.].

FERONIA Confers Resistance to Photooxidative Stress in Arabidopsis.

Plants absorb light energy required for photosynthesis, but excess light can damage plant cells. To protect themselves, plants have developed diverse signaling pathways which are activated under high-intensity light. Plant photoprotection mechanisms have been mainly investigated under conditions of extremely high amount of light; thus, it is largely unknown how plants manage photooxidative damage under moderate light intensities. In the present study, we found that FERONIA (FER) is a key protein that confers resistance to photooxidative stress in plants under moderate light intensity. FER-deficient mutants were highly susceptible to increasing light intensity and exhibited photobleaching even under moderately elevated light intensity (ML). Light-induced expression of stress genes was largely diminished by the fer-4 mutation. In addition, excitation pressure on Photosystem II was significantly increased in fer-4 mutants under ML. Consistently, reactive oxygen species, particularly singlet oxygen, accumulated in fer-4 mutants grown under ML. FER protein abundance was found to be elevated after exposure to ML, which is indirectly affected by the ubiquitin-proteasome pathway. Altogether, our findings showed that plants require FER-mediated photoprotection to maintain their photosystems even under moderate light intensity.