| 1. |
- Andreasson, Erik, et al.
(författare)
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Utilization of protoplasts to facilitate gene editing in plants: schemes for in vitro shoot regeneration from tissues and protoplasts of potato and rapeseed: implications of bioengineering such as gene editing of broad-leaved plants
- 2022
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Ingår i: Frontiers in Genome Editing. - : Frontiers Media SA. - 2673-3439. ; 4
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Forskningsöversikt (refereegranskat)abstract
- Schemes for efficient regeneration and recovery of shoots from in vitro tissues or single cells, such as protoplasts, are only available for limited numbers of plant species and genotypes and are crucial for establishing gene editing tools on a broader scale in agriculture and plant biology. Growth conditions, including hormone and nutrient composition as well as light regimes in key steps of known regeneration protocols, display significant variations, even between the genotypes within the same species, e.g., potato (Solanum tuberosum). As fresh plant material is a prerequisite for successful shoot regeneration, the plant material often needs to be refreshed for optimizing the growth and physiological state prior to genetic transformation. Utilization of protoplasts has become a more important approach for obtaining transgene-free edited plants by genome editing, CRISPR/Cas9. In this approach, callus formation from protoplasts is induced by one set of hormones, followed by organogenesis, i.e., shoot formation, which is induced by a second set of hormones. The requirements on culture conditions at these key steps vary considerably between the species and genotypes, which often require quantitative adjustments of medium compositions. In this mini-review, we outline the protocols and notes for clonal regeneration and cultivation from single cells, particularly protoplasts in potato and rapeseed. We focus mainly on different hormone treatment schemes and highlight the importance of medium compositions, e.g., sugar, nutrient, and light regimes as well as culture durations at the key regeneration steps. We believe that this review would provide important information and hints for establishing efficient regeneration strategies from other closely related and broad-leaved plant species in general.
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| 2. |
- Bubolz, Jéssica, et al.
(författare)
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Genetically modified (GM) late blight-resistant potato and consumer attitudes before and after a field visit
- 2022
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Ingår i: GM Crops and Food Biotechnology in Agriculture and the Food Chain. - : Informa UK Limited. - 2164-5698 .- 2164-5701. ; 13, s. 290-298
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Tidskriftsartikel (refereegranskat)abstract
- Late blight, caused by Phytophthora infestans, is the most devastating disease in potato production. Here, we show full late blight resistance in a location with a genetically diverse pathogen population with the use of GM potato stacked with three resistance (R) genes over three seasons. In addition, using this field trials, we demonstrate that in-the-field intervention among consumers led to change for more favorable attitude generally toward GM crops.
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| 3. |
- Iqbal, Mudassir, et al.
(författare)
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Biological control of strawberry crown rot, root rot and grey mould by the beneficial fungus Aureobasidium pullulans
- 2021
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Ingår i: BioControl. - : Springer Science and Business Media LLC. - 1386-6141 .- 1573-8248. ; 66, s. 535-545
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Tidskriftsartikel (refereegranskat)abstract
- Utilization of biocontrol agents is a sustainable approach to reduce plant diseases caused by fungal pathogens. In the present study, we tested the effect of the candidate biocontrol fungus Aureobasidium pullulans (De Bary) G. Armaud on strawberry under in vitro and in vivo conditions to control crown rot, root rot and grey mould caused by Phytophthora cactorum (Lebert and Cohn) and Botrytis cinerea Pers, respectively. A dual plate confrontation assay showed that mycelial growth of P. cactorum and B. cinerea was reduced by 33-48% when challenged by A. pullulans as compared with control treatments. Likewise, detached leaf and fruit assays showed that A. pullulans significantly reduced necrotic lesion size on leaves and disease severity on fruits caused by P. cactorum and B. cinerea. In addition, greenhouse experiments with whole plants revealed enhanced biocontrol efficacy against root rot and grey mould when treated with A. pullulans either in combination with the pathogen or pre-treated with A. pullulans followed by inoculation of the pathogens. Our results demonstrate that A. pullulans is an effective biocontrol agent to control strawberry diseases caused by fungal pathogens and can be an effective alternative to chemical-based fungicides.
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| 4. |
- Kalyandurg, Pruthvi Balachandra, et al.
(författare)
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Spray-Induced Gene Silencing as a Potential Tool to Control Potato Late Blight Disease
- 2021
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Ingår i: Phytopathology. - 0031-949X .- 1943-7684. ; 111, s. 2168-2175
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Tidskriftsartikel (refereegranskat)abstract
- Phytophthora infestans causes late blight disease on potato and tomato and is currently controlled by resistant cultivars or intensive fungicide spraying. Here, we investigated an alternative means for late blight control by spraying potato leaves with double-stranded RNAs (dsRNA) that target the P. infestans genes essential for infection. First, we showed that the sporangia of P. infestans expressing green fluorescent protein (GFP) can take up in vitro synthesized dsRNAs homologous to GFP directly from their surroundings, including leaves, which led to the reduced relative expression of GFP. We further demonstrate the potential of spray-induced gene silencing (SIGS) in controlling potato late blight disease by targeting developmentally important genes in P. infestans such as guanine-nucleotide binding protein beta-subunit (PiGPB1), haustorial membrane protein (PiHmp1), cutinase (PiCut3), and endo-1,3(4)-beta-glucanase (PiEndo3). Our results demonstrate that SIGS can potentially be used to mitigate potato late blight; however, the degree of disease control is dependent on the selection of the target genes.
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| 5. |
- Karlsson, Milla, et al.
(författare)
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CRISPR/Cas9 genome editing of potato StDMR6-1 results in plants less affected by different stress conditions
- 2024
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Ingår i: Horticulture Research. - 2052-7276. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Potato is the third most important food crop, but cultivation is challenged by numerous diseases and adverse abiotic conditions. To combat diseases, frequent fungicide application is common. Knocking out susceptibility genes by genome editing could be a durable option to increase resistance. DMR6 has been described as a susceptibility gene in several crops, based on data that indicates increased resistance upon interruption of the gene function. In potato, Stdmr6-1 mutants have been described to have increased resistance against the late blight pathogen Phytophthora infestans in controlled conditions. Here, we present field evaluations of CRISPR/Cas9 mutants, in a location with a complex population of P. infestans, during four consecutive years that indicate increased resistance to late blight without any trade-off in terms of yield penalty or tuber quality. Furthermore, studies of potato tubers from the field trials indicated increased resistance to common scab, and the mutant lines exhibit increased resistance to early blight pathogen Alternaria solani in controlled conditions. Early blight and common scab are problematic targets in potato resistance breeding, as resistance genes are very scarce. The described broad-spectrum resistance of Stdmr6-1 mutants may further extend to some abiotic stress conditions. In controlled experiments of either drought simulation or salinity, Stdmr6-1 mutant plants are less affected than the background cultivar. Together, these results demonstrate the prospect of the Stdmr6-1 mutants as a useful tool in future sustainable potato cultivation without any apparent trade-offs.
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| 6. |
- Resjö, Svante, et al.
(författare)
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Proteomics of PTI and Two ETI Immune Reactions in Potato Leaves
- 2019
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067 .- 1661-6596. ; 20:19
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Tidskriftsartikel (refereegranskat)abstract
- Plants have a variety of ways to defend themselves against pathogens. A commonly used model of the plant immune system is divided into a general response triggered by pathogen-associated molecular patterns (PAMPs), and a specific response triggered by effectors. The first type of response is known as PAMP triggered immunity (PTI), and the second is known as effector-triggered immunity (ETI). To obtain better insight into changes of protein abundance in immunity reactions, we performed a comparative proteomic analysis of a PTI and two different ETI models (relating to Phytophthora infestans) in potato. Several proteins showed higher abundance in all immune reactions, such as a protein annotated as sterol carrier protein 2 that could be interesting since Phytophthora species are sterol auxotrophs. RNA binding proteins also showed altered abundance in the different immune reactions. Furthermore, we identified some PTI-specific changes of protein abundance, such as for example, a glyoxysomal fatty acid beta-oxidation multifunctional protein and a MAR-binding protein. Interestingly, a lysine histone demethylase was decreased in PTI, and that prompted us to also analyze protein methylation in our datasets. The proteins upregulated explicitly in ETI included several catalases. Few proteins were regulated in only one of the ETI interactions. For example, histones were only downregulated in the ETI-Avr2 interaction, and a putative multiprotein bridging factor was only upregulated in the ETI-IpiO interaction. One example of a methylated protein that increased in the ETI interactions was a serine hydroxymethyltransferase.
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| 7. |
- Zahid, Muhammad Awais, et al.
(författare)
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A fast, nondestructive method for the detection of disease-related lesions and wounded leaves
- 2021
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Ingår i: Biotechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 71
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Tidskriftsartikel (refereegranskat)abstract
- Trypan blue staining is a classic way of visualizing leaf disease and wound responses in plants, but it involves working with toxic chemicals and is time-consuming (2-3 days). Here, the investigators established near-infrared scanning with standard lab equipment as a fast and nondestructive method for the analysis of leaf injuries compared with trypan blue staining. Pathogen-inoculated and wounded leaves from potato, tomato, spinach, strawberry, and arabidopsis plants were used for proof of concept. The results showed that this newly developed protocol with near-infrared scanning gave the same results as trypan blue staining. Furthermore, a macro in FIJI was made to quantify the leaf damage. The new protocol was time-efficient, nondestructive, chemical-free and may be used for high-throughput studies.
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| 8. |
- Zahid, Muhammad Awais, et al.
(författare)
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A Quantitative Luminol-Based Assay for ROS Burst Detection in Potato Leaves in Response to Biotic Stimuli
- 2022
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Ingår i: Plant Pathology : Method and Protocols. - New York, NY : Springer US. - 9781071625163 ; 2536, s. 395-402
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Bokkapitel (refereegranskat)abstract
- Reactive oxygen species (ROS) are important signaling agents in plants and animals. They are involved in diverse processes, including activation of immune responses to pathogen infection. Biphasic detection of ROS in response to pathogen perception is becoming more popular even in important crops like potato as means of screening different germ plasms and mutants generated by for example CRISPR-Cas9 as well as identifying signaling pathways. Here we describe a detailed protocol for quantifying ROS bursts induced in potato leaf discs in response to a bacterial elicitor and Phytophthora infestans.
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| 9. |
- Zahid, Muhammad Awais, et al.
(författare)
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Enhanced stress resilience in potato by deletion of Parakletos
- 2024
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Ingår i: Nature Communications. - 2041-1723. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Continued climate change impose multiple stressors on crops, including pathogens, salt, and drought, severely impacting agricultural productivity. Innovative solutions are necessary to develop resilient crops. Here, using quantitative potato proteomics, we identify Parakletos, a thylakoid protein that contributes to disease susceptibility. We show that knockout or silencing of Parakletos enhances resistance to oomycete, fungi, bacteria, salt, and drought, whereas its overexpression reduces resistance. In response to biotic stimuli, Parakletos-overexpressing plants exhibit reduced amplitude of reactive oxygen species and Ca2+ signalling, and silencing Parakletos does the opposite. Parakletos homologues have been identified in all major crops. Consecutive years of field trials demonstrate that Parakletos deletion enhances resistance to Phytophthora infestans and increases yield. These findings demark a susceptibility gene, which can be exploited to enhance crop resilience towards abiotic and biotic stresses in a low-input agriculture.
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| 10. |
- Zahid, Muhammad Awais
(författare)
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Insights into potato plant immunity reveals Parakletos as a novel ROS suppressor
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Potato (Solanum tuberosum L.) is the world's third most commonly grown food crop with very high yield potential. However, its production is hampered by several pathogens, with consequent yield losses. Existing control methods include frequent, costly fungicide applications, and classical breeding is complicated in potato. One way to develop new longterm solutions is to improve understanding of plant molecular immunity and factors capable of providing broad-spectrum resistance. To assist such efforts, comparative proteomic techniques were applied to enhance understanding of changes in protein abundance during the immune responses of potato. These focused on a PTI (Pattern-triggered immunity) model and two ETI (Effector-triggered immunity) models (both related to resistance genes to Phytophthora infestans, the causal agent of late blight) in potato to enhance understanding of changes in protein abundance during its immune responses. Numerous proteins increased in abundance in all observed immune responses, including one identified as sterol transporter protein 2, which is intriguing because sterol content plays an important role in plant immunity, and oomycete pathogens like P. infestans rely on their hosts for sterols. The abundance of RNA binding proteins also changed in different immune reactions. A few proteins changed in abundance in only one of the ETI models, e.g., histones were downregulated in one (ETI responses to P. infestans effector Avr2), whereas a putative multiprotein bridging factor was upregulated in the other (ETI responses to P. infestans effector IpiO). Intriguingly, the proteomic differences between the two ETI models were of similar magnitude to those between the ETI models and PTI. The next step was to study the general PTI-related defence potato proteome by two-step fractionation and new bioinformatics analyses. Five candidates with potential importance in our proteomic dataset were selected for functional validation studies. To facilitate validation studies, a luminol-based assay was developed to study biphasic reactive oxygen species (ROS) bursts in potato leaves induced by flg22 and P. infestans as well as an easy, cost-effective method using near-IR scanning to quantify leaf wounding and disease lesion areas without damaging leaves. Over-expression and silencing of one identified protein, named Parakletos, respectively increased and reduced P. infestans infection in Nicotiana benthamiana. Moreover, its overexpression suppressed the ROS burst response to flg22, while its silencing increased it. Transcript analyses showed upregulation of defence-related genes (e.g., ICS1, PR1, PTI5, and RBOHB) in response to flg22 in Parakletos-silenced plants. Expression of light-harvesting complex B6 (LHCB6) was also enhanced in plants overexpressing Parakletos. It was found that Parakletos co-localized with the Calcium Sensing Receptor (CAS) in chloroplasts, and that it is functionally dependent on CAS. Furthermore, CRISPR/Cas9-mediated knock-out (KO) of parakletos in potato enhanced broad-spectrum resistance to late and early blight in controlled condition. It also reduced infection to Pseudomonas syringae in N. benthamiana. Moreover, during field trials the parakletos-KO lines showed enhanced resistance to P. infestans. These findings contribute to our understanding of plant immunity and provide a new susceptibility-gene based strategy for building broad-spectrum disease resistance in crops.
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