CRISPR/Cas9 genome editing of potato StDMR6-1 results in plants less affected by different stress conditions.

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.

Enhanced stress resilience in potato by deletion of Parakletos.

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.

Genetically modified (GM) late blight-resistant potato and consumer attitudes before and after a field visit.

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.

Invited Mini-Review Research Topic: 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.

Schemes for efficient regenerationand 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.

A Quantitative Luminol-Based Assay for ROS Burst Detection in Potato Leaves in Response to Biotic Stimuli.

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.

A fast, nondestructive method for the detection of disease-related lesions and wounded leaves.

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.

Spray-Induced Gene Silencing as a Potential Tool to Control Potato Late Blight Disease.

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 ß-subunit (PiGPB1), haustorial membrane protein (PiHmp1), cutinase (PiCut3), and endo-1,3(4)-ß-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.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Proteomics of PTI and Two ETI Immune Reactions in Potato Leaves.

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.

Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana.

Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α-Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose-dependent. Activity-based protein profiling (ABPP) revealed that the activities of papain-like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.

Plant Growth Promotion and Suppression of Bacterial Leaf Blight in Rice by Inoculated Bacteria.

The present study was conducted to evaluate the potential of rice rhizosphere associated antagonistic bacteria for growth promotion and disease suppression of bacterial leaf blight (BLB). A total of 811 rhizospheric bacteria were isolated and screened against 3 prevalent strains of BLB pathogen Xanthomonas oryzae pv. oryzae (Xoo) of which five antagonistic bacteria, i.e., Pseudomonas spp. E227, E233, Rh323, Serratia sp. Rh269 and Bacillus sp. Rh219 showed antagonistic potential (zone of inhibition 1-19 mm). Production of siderophores was found to be the common biocontrol determinant and all the strains solubilized inorganic phosphate (82-116 µg mL-1) and produced indole acetic acid (0.48-1.85 mg L-1) in vitro. All antagonistic bacteria were non-pathogenic to rice, and their co-inoculation significantly improved plant health in terms of reduced diseased leaf area (80%), improved shoot length (31%), root length (41%) and plant dry weight (60%) as compared to infected control plants. Furthermore, under pathogen pressure, bacterial inoculation resulted in increased activity of defense related enzymes including phenylalanine ammonia-lyase and polyphenol oxidase, along with 86% increase in peroxidase and 53% increase in catalase enzyme activities in plants inoculated with Pseudomonas sp. Rh323 as well as co-inoculated plants. Bacterial strains showed good colonization potential in the rice rhizosphere up to 21 days after seed inoculation. Application of bacterial consortia in the field resulted in an increase of 31% in grain yield and 10% in straw yield over non-inoculated plots. Although, yield increase was statistically non-significant but was accomplished with overall saving of 20% chemical fertilizers. The study showed that Pseudomonas sp. Rh323 can be used to develop dual-purpose inoculum which can serve not only to suppress BLB but also to promote plant growth in rice.