Opportunities and Challenges of In Vitro Tissue Culture Systems in the Era of Crop Genome Editing.

Currently, the development of genome editing (GE) tools has provided a wide platform for targeted modification of plant genomes. However, the lack of versatile DNA delivery systems for a large variety of crop species has been the main bottleneck for improving crops with beneficial traits. Currently, the generation of plants with heritable mutations induced by GE tools mostly goes through tissue culture. Unfortunately, current tissue culture systems restrict successful results to only a limited number of plant species and genotypes. In order to release the full potential of the GE tools, procedures need to be species and genotype independent. This review provides an in-depth summary and insights into the various in vitro tissue culture systems used for GE in the economically important crops barley, wheat, rice, sorghum, soybean, maize, potatoes, cassava, and millet and uncovers new opportunities and challenges of already-established tissue culture platforms for GE in the crops.

New Hope for Genome Editing in Cultivated Grasses: CRISPR Variants and Application.

With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) mediated genome editing, crop improvement has progressed significantly in recent years. In this genome editing tool, CRISPR-associated Cas nucleases are restricted to their target of DNA by their preferred protospacer adjacent motifs (PAMs). A number of CRISPR-Cas variants have been developed e.g. CRISPR-Cas9, -Cas12a and -Cas12b, with different PAM requirements. In this mini-review, we briefly explain the components of the CRISPR-based genome editing tool for crop improvement. Moreover, we intend to highlight the information on the latest development and breakthrough in CRISPR technology, with a focus on a comparison of major variants (CRISPR-Cas9, -Cas12a, and -Cas12b) to the newly developed CRISPR-SpRY that have nearly PAM-less genome editing ability. Additionally, we briefly explain the application of CRISPR technology in the improvement of cultivated grasses with regard to biotic and abiotic stress tolerance as well as improving the quality and yield.

Barley Nepenthesin-Like Aspartic Protease HvNEP-1 Degrades Fusarium Phytase, Impairs Toxin Production, and Suppresses the Fungal Growth.

Nepenthesins are categorized under the subfamily of the nepenthesin-like plant aspartic proteases (PAPs) that form a distinct group of atypical PAPs. This study describes the effect of nepenthesin 1 (HvNEP-1) protease from barley (Hordeum vulgare L.) on fungal histidine acid phosphatase (HAP) phytase activity. Signal peptide lacking HvNEP-1 was expressed in Pichia pastoris and biochemically characterized. Recombinant HvNEP-1 (rHvNEP-1) strongly inhibited the activity of Aspergillus and Fusarium phytases, which are enzymes that release inorganic phosphorous from phytic acid. Moreover, rHvNEP-1 suppressed in vitro fungal growth and strongly reduced the production of mycotoxin, 15-acetyldeoxynivalenol (15-ADON), from Fusarium graminearum. The quantitative PCR analysis of trichothecene biosynthesis genes (TRI) confirmed that rHvNEP-1 strongly repressed the expression of TRI4, TRI5, TRI6, and TRI12 in F. graminearum. The co-incubation of rHvNEP-1 with recombinant F. graminearum (rFgPHY1) and Fusarium culmorum (FcPHY1) phytases induced substantial degradation of both Fusarium phytases, indicating that HvNEP-1-mediated proteolysis of the fungal phytases contributes to the HvNEP-1-based suppression of Fusarium.

Molecular Properties and New Potentials of Plant Nepenthesins.

Nepenthesins are aspartic proteases (APs) categorized under the A1B subfamily. Due to nepenthesin-specific sequence features, the A1B subfamily is also named nepenthesin-type aspartic proteases (NEPs). Nepenthesins are mostly known from the pitcher fluid of the carnivorous plant Nepenthes, where they are availed for the hydrolyzation of insect protein required for the assimilation of insect nitrogen resources. However, nepenthesins are widely distributed within the plant kingdom and play significant roles in plant species other than Nepenthes. Although they have received limited attention when compared to other members of the subfamily, current data indicates that they have exceptional molecular and biochemical properties and new potentials as fungal-resistance genes. In the current review, we provide insights into the current knowledge on the molecular and biochemical properties of plant nepenthesins and highlights that future focus on them may have strong potentials for industrial applications and crop trait improvement.

Aspergillus ficuum phytase activity is inhibited by cereal grain components.

In the current study, we report for the first time that grain components of barley, rice, wheat and maize can inhibit the activity of Aspergillus ficuum phytase. The phytase inhibition is dose dependent and varies significantly between cereal species, between cultivars of barley and cultivars of wheat and between Fusarium graminearum infected and non-infected wheat grains. The highest endpoint level of phytase activity inhibition was 90%, observed with grain protein extracts (GPE) from F. graminearum infected wheat. Wheat GPE from grains infected with F. graminearum inhibits phytase activity significantly more than GPE from non-infected grains. For four barley cultivars studied, the IC50 value ranged from 0.978 ± 0.271 to 3.616 ± 0.087 mg×ml-1. For two non-infected wheat cultivars investigated, the IC50 values were varying from 2.478 ± 0.114 to 3.038 ± 0.097 mg×ml-1. The maize and rice cultivars tested gaveIC50 values on 0.983 ± 0.205 and 1.972 ± 0.019 mg×ml-1, respectively. After purifying the inhibitor from barley grains via Superdex G200, an approximately 30-35 kDa protein was identified. No clear trend for the mechanism of inhibition could be identified via Michaelis-Menten kinetics and Lineweaver-Burk plots. However, testing of the purified phytase inhibitor together with the A. ficuum phytase and the specific protease inhibitors pepstatin A, E64, EDTA and PMSF revealed that pepstatin A repealed the phytase inhibition. This indicates that the observed inhibition of A. ficuum phytase by cereal grain extracts is caused by protease activity of the aspartic proteinase type.