Breeding rice for yield improvement through CRISPR/Cas9 genome editing method: current technologies and examples.

The impending climate change is threatening the rice productivity of the Asian subcontinent as instances of crop failures due to adverse abiotic and biotic stress factors are becoming common occurrences. CRISPR-Cas9 mediated genome editing offers a potential solution for improving rice yield as well as its stress adaptation. This technology allows modification of plant's genetic elements and is not dependent on foreign DNA/gene insertion for incorporating a particular trait. In this review, we have discussed various CRISPR-Cas9 mediated genome editing tools for gene knockout, gene knock-in, simultaneously disrupting multiple genes by multiplexing, base editing and prime editing the genes. The review here also presents how these genome editing technologies have been employed to improve rice productivity by directly targeting the yield related genes or by indirectly manipulating various abiotic and biotic stress responsive genes. Lately, many countries treat genome-edited crops as non-GMOs because of the absence of foreign DNA in the final product. Thus, genome edited rice plants with improved yield attributes and stress resilience are expected to be accepted by the public and solve food crisis of a major portion of the globe. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01423-y.

A simplified and improved protocol of rice transformation to cater wide range of rice cultivars.

The latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efficiencies which necessities screening of large number of transgenic plants to find the suitable edits. In the present study, we have simplified the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76-78 days. We also obtained about 90% regeneration efficiency for both Japonica and Indica cultivars. The transformation efficiency was about 97% in the case of Japonica and 69-83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efficiency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efficiency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars.

Application of Copper-Chitosan Nanoparticles Stimulate Growth and Induce Resistance in Finger Millet (Eleusine coracana Gaertn.) Plants against Blast Disease.

Copper-chitosan nanoparticle (CuChNp) was synthesized and used to study its effect on finger millet plant as a model plant system. Our objective was to explore the efficacy of CuChNp application to control blast disease of finger millet. CuChNp was applied to finger millet either as a foliar spray or as a combined application (involving seed coat and foliar spray). Both the application methods enhanced growth profile of finger millet plants and increased yield. The increased yield was nearly 89% in combined application method. Treated finger millet plants challenged with Pyricularia grisea showed suppression of blast disease development when compared to control. Nearly 75% protection was observed in the combined application of CuChNp to finger millet plants. In CuChNp treated finger millet plants, a significant increase in defense enzymes was observed, which was detected both qualitatively and quantitatively. The suppression of blast disease correlates well with increased defense enzymes in CuChNp treated finger millet plants.

Withanolide production by fungal endophyte isolated from Withania somnifera.

Withanolides, the secondary metabolite from Withania species is used in Ayurvedic medicine and now proved to have potential use in treating cardiovascular, Alzheimer's disease, etc. Its production in plants varies between genotypes with very low yield. For improved industrial commercialisation, there is a need to increase its production. Endophytic fungi are symbiotically associated with plants and can synthesise the same bioactive compounds and natural products as their host plant. There are no reports available on withanolide-producing (endophytic) fungi. The present study identified an endophytic fungus (Taleromyces pinophilus) from leaves of Withania somnifera which produces withanolides in the medium. The structure of withanolide was confirmed by 1H NMR, LC-MS analyses and quantified by HPLC analysis. The fungus produces high amount of withanolide when compared to leaf and root of W. somnifera. The fungus can be exploited to produce the withanolide to meet its demand.

Chitosan nanoparticle induced defense responses in fingermillet plants against blast disease caused by Pyricularia grisea (Cke.) Sacc.

The in vitro antifungal properties of chitosan nanoparticle and its role in protection of fingermillet plants from blast disease were evaluated. Chitosan nanoparticle inhibited the radial growth of Pyricularia grisea indicating the antifungal property. Application of chitosan nanoparticle delayed blast symptom expression on fingermillet leaves for 25days while it was on 15day in control plants. Chitosan naoparticle was able to induce the reactive oxygen species and the level of peroxidase actvitiy in leaves of fingermillet, which might be the reason for delayed symptom. The treated plants showed reduced disease incidence when compared to untreated control plants. These results suggested the role of chitosan nanoparticle in protecting fingermillet plants from P. grisea infection.

Isolation, characterization and molecular three-dimensional structural predictions of metalloprotease from a phytopathogenic fungus, Alternaria solani (Ell. and Mart.) Sor.

The present study aims at isolation, identification, characterization and prediction of three-dimensional molecular architecture of a proteolytic enzyme from the early blight pathogen, Alternaria solani which are hypothesized to be a marker of phytopathogenicity. Maximum enzyme production by A. solani was observed in Czapex's Dox broth amended with 2% (w/v) casein than other inducer amendments. Results indicate that the enzyme remained highly active in a pH range of 7.0-10.0 and a temperature range of 45-50°C. The enzyme was strongly inhibited by EDTA, whereas phenylmethylsulfonyl fluoride and monovalent cations (Na(+), K(+)) had little effect. Metal ions such as MgSO4, CaCl2, KCl at 10 mM concentration showed a stimulatory effect (>85%) on protease activity. Matrix-assisted laser desorption and ionization time of flight/mass spectrometry analysis of partially purified enzyme revealed the presence of protease belonging to a keratinolytic protein (metalloprotease) of exopeptidase nature. Putative A. solani keratinolytic enzyme (AsK) is made up of 216 amino acid residues with molecular weight (MW) 24.5 kDa, having a molecular formula of C1094H1704N290O342S4. Ramachandran plot analysis of the protein residues falling into the most favored secondary structures was observed at 84.2%. The major protein structural blocks, 2-ß-sheets, and 9-α-helices have a greater tendency to be conserved during the evolutionary process than do mere sequences of amino acids. Besides, AsK, model prediction showed the presence of a Zinc atom at helix regions (Helix 3, 6, 7: His(57), His(130), His(169), and Cys(123)). Thus, it can be concluded that the major proteinases of AsK are divalent cation-requiring metalloproteinases and make them potential targets of protease inhibitors designing.

Preparation of Chitosan nanoparticles and its effect on detached rice leaves infected with Pyricularia grisea.

The aim of the present study was to prepare chitosan nanoparticles to evaluate their effect on protection of rice plants from blast fungus. Nanoparticles were prepared using the ionic gelation method by the interaction of Chitosan and sodium tripolyphosphate. The particle size, polydispersity index, zetapotential and structure was confirmed by DLS, FTIR, TEM and XRD. The Chitosan nanoparticle was evaluated for suppression of rice blast fungus (Pyricularia grisea) under the detached leaf condition. It is evident from our results that chitosan nanoparticle have potential in suppressing blast disease of rice which can be used further under field condition to protect rice plants from the devastating fungus.

Screening of different Trichoderma species against agriculturally important foliar plant pathogens.

Different isolates of Trichoderma were isolated from soil samples which were collected from different part of India. These isolates were grouped into four Trichoderma species viz., Trichoderma asperellum (Ta), T. harzianum (Th), T. pseudokoningii (Tp) and T. longibrachiatum (Tl) based on their morphological characters. Identification of the above isolates was also confirmed through ITS region analysis. These Trichoderma isolates were tested for in vitro biological control of Alternaria solani, Bipolaris oryzae, Pyricularia oryzae and Sclerotinia scierotiorum which cause serious diseases like early blight (target spot) of tomato and potato, brown leaf spot disease in rice, rice blast disease, and white mold disease in different plants. Under in vitro conditions, all the four species of Trichoderma (10 isolates) proved 100% potential inhibition against rice blast pathogen Pyracularia oryzae. T. harzianum (Th-01) and T. asperellum (Ta-10) were effective with 86.6% and 97.7%, growth inhibition of B. oryzae, respectively. Among others, T. pseudokoningii (Tp-08) and T. Iongibrachiatum (Tl-09) species were particularly efficient in inhibiting growth of S. sclerotiorum by 97.8% and 93.3%. T. Iongibrachiatum (TI-06 and TI-07) inhibited maximum mycelial growth of A. solani by 87.6% and 84.75. However, all the T. harzianum isolates showed significantly higher inhibition against S. sclerotiorum (CD value 9.430), causing white mold disease. This study led to the selection of potential Trichoderma isolates against rice blast, early blight, brown leaf spot in rice and white mold disease in different crops.

Protection of turmeric plants from rhizome rot disease under field conditions by ß-D-glucan nanoparticle.

The rhizome rot caused by Pythium aphanidermatum is one of the most devastating diseases of the turmeric crop. Fungicides are unable to control the rapidly evolving P. aphanidermatum and new control strategies are urgently needed. This study examined the effect of ß-d-glucan nanoparticles (GNP) in turmeric plants under field condition by the foliar spray method. Enhanced plant growth, rhizome yield, and curcumin content demonstrate the positive effect of the GNP on turmeric plants. Rapid activation of various defense enzymes was also observed in leaves and rhizomes of treated plants. GNP-treated plants showed a decreased rot incidence. It may be possible that increased defense enzymes might have played a role in reducing the colonization of pathogen.

Lovastatin-producing endophytic fungus isolated from a medicinal plant Solanum xanthocarpum.

Lovastatin is a potent drug for lowering blood cholesterol. An endophytic fungus Phomopsis vexans was isolated from the healthy leaf tissues of Solanum xanthocarpum, a medicinal plant, and screened for lovastatin production. The fungus was identified by their characteristic cultural morphology and molecular analysis. The strain had a component with the same TLC Rf value and HPLC retention time as authentic lovastatin. The presence of lovastatin was further confirmed by FT-IR, UV, (1)H, (13)C NMR and LC-MS analyses. The amount of lovastatin produced by this endophytic fungus was quantified to be 550 mg/L, and thus the fungus can serve as a potential material to improve the production of lovastatin.

Foliar application of ß-D-glucan nanoparticles to control rhizome rot disease of turmeric.

The soilborne Oomycete Pythium aphanidermatum is the causal agent of rhizome rot disease, one of the most serious threats to turmeric crops. At present, effective fungicides are not available. Researches on nanoparticles in a number of crops have evidenced the positive changes in gene expression indicating their potential use in crop improvement. Hence, experiments were carried out to determine the effect of ß-D-glucan nanoparticles (nanobiopolymer) in protection of turmeric plants against rot disease by the way of products that reinforce plant's own defense mechanism. Foliar spray of ß-D-glucan nanoparticles (0.1%, w/v) elicited marked increase in the activity of defense enzymes such as peroxidases (E.C.1.11.1.7), polyphenol oxidases (E.C.1.14.18.1), protease inhibitors (E.C.3.4.21.1) and ß-1,3-glucanases (E.C.3.2.1.39) at various age levels. Constitutive and induced isoforms of these enzymes were investigated during this time-course study. ß-D-glucan nanoparticles (GNPs) significantly reduced the rot incidence offering 77% protection. Increased activities of defense enzymes in GNPs-applied turmeric plants may play a role in restricting the development of disease symptoms. These results demonstrated that GNPs could be used as an effective resistance activator in turmeric for control of rhizome rot disease.

ß-D-Glucan nanoparticle pre-treatment induce resistance against Pythium aphanidermatum infection in turmeric.

In vitro experiments were carried out to test the efficacy of GNP (ß-D-glucan nanoparticle prepared from mycelium of Pythium aphanidermatum) against rhizome rot disease of turmeric (Curcuma longa L.) caused by P. aphanidermatum. GNP (0.1%, w/v) was applied to rhizome prior to inoculation with P. aphanidermatum (0 h, 24 h). Cell death, activities of defense enzymes such as peroxidase, polyphenol oxidase, protease inhibitor and ß-1,3 glucanase were monitored. Prior application of GNP (24 h) to turmeric rhizome effectively controls P. aphanidermatum infection. The increase in defense enzyme activities occurred more rapidly and was enhanced in P. aphanidermatum infected rhizomes that were pre-treated with GNP. Pre-treatment also induced new isoforms of defense enzymes. Increased activities of defense enzymes suggest that they play a key role in restricting the development of disease symptoms in the rhizomes as evidenced by a reduction in cell death. The results demonstrated that GNP can be used as a potential agent for control of rhizome rot disease.

Preparation of ß-D-glucan nanoparticles and its antifungal activity.

This work demonstrates the preparation of ß-D-glucan (isolated from the cell wall of Pythium aphanidermatum) nanoparticles through the addition of 2% (w/v) sodium hydroxide to ß-D-glucan solution with constant stirring at 90°C. Addition of sodium tripolyphosphate (TPP) aids the stable formation of nanoparticles. Fourier transform infrared (FTIR) spectroscopy confirmed phosphoric groups of TPP linked with OH group of ß-D-glucan in the nanoparticles. The formation of nanoparticles was observed by the peak at 386 nm using UV-vis spectroscopy. The average size of nanoparticle as determined by Zetasizer was about 60 nm, while the zeta potential was negative. Scanning electron microscope image showed spherical, smooth and almost homogenous structure for nanoparticles with size ranging from 20 to 50 nm. Further analysis by TEM, indicated polydispersity with an average size of 20-30 nm. The XRD analysis confirmed the crystalline structure of ß-glucan nanoparticle. The prepared nanoparticles exhibited antifungal activity against P. aphanidermatum, a devastating fungus which affects major crop plants.

Gymnemagenin-producing endophytic fungus isolated from a medicinal plant Gymnema sylvestre R.Br.

Gymnema sylvestre is a plant containing the triterpenoid gymnemagenin, which is used in the pharmaceutical industry as an antidiabetic agent. The objective of this study was to determine whether endophytic fungi, isolated from G. sylvestre, produce gymnemagenin. We isolated an endophytic fungal strain from the leaves of G. sylvestre which produces gymnemagenin in the medium. The fungus was identified as Penicillium oxalicum based on morphological and molecular methods. The strain had a component with the same TLC Rf value and HPLC retention time as authentic gymnemagenin. The presence of gymnemagenin was further confirmed by FTIR, UV, and (1)H NMR analyses.

Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus, Alternaria solani.

Serine proteases are involved in an enormous number of biological processes. The present study aims at characterizing three-dimensional (3D) molecular architecture of serine proteases from early blight pathogen, Alternaria solani that are hypothesized to be markers of phytopathogenicity. A serine protease was purified to homogeneity and MALDI-TOF-MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases (AsP). AsP is made up of 403 amino acid residues with molecular weight of 42.1 kDa (Isoelectric point - 6.51) and its molecular formula was C1859 H2930 N516 O595 S4 . AsP structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 ß-sheets and 10 α-helices, with Ser(350) (G347-G357), Asp(158) (D158-H169), and His(193) (H193-G203) in separate turn/coil structures. Biological metal binding region situated near 6th-helix and His(193) residue is responsible for metal binding site. Also, calcium ion (Ca(2+)) is coordinated by the carboxyl groups of Lys(84), Ile(85), Lys(86), Asp(87), Phe(88), Ala(89), Ala(90) (K84-A90) for first Ca(2+) binding site and carbonyl oxygen atom of Lys(244), Gly(245), Arg(246), Thr(247), Lys(248), Lys(249), and Ala(250) (K244-A250), for second Ca(2+) binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT, and VADAR servers to confirm the geometry and stereo-chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing.

Production, partial purification and characterization of protease from a phytopathogenic fungi Alternaria solani (Ell. and Mart.) Sorauer.

An alkaline serine protease producing strain Alternaria solani was optimized for its enzyme production under submerged conditions. The maximum production of protease by A. solani was achieved by using sodium nitrate at the optimum concentration of 0.2% w/v. A. solani produced higher quantities (3.75 [unit/mg of protein]) of an inducible extracellular proteases on day 9 after incubation in czapek's dox broth medium amended with 1% casein as an inducer at pH 8.5, temperature 27 °C and 3% sucrose as carbon source. Extracellular proteases were precipitated by ammonium sulphate saturation (80%) method and purified on Sephadex G-100 column chromatography. The molecular mass of SDS-PAGE and Sephadex G-100 Column Gel permeation chromatography purified protease was estimated to 42 kDa. In addition, trypsin digestion of 42 kDa protein band was carried out and analyzed by MALDI-TOF for the identification of protease. The sequence IKELATNGVVTNVK (378-391) segment of the alkaline serine protease was found by using MS/MS spectrum at 1485 m/z from the purified fraction. It showed optimal activity at 50 °C and pH 9-10 and broad pH stability between pH 6-12. The protease activity was inhibited by phenyl methyl sulfonyl fluoride (PMSF), all the results indicated that the presence of a serine residue in the active site and is thus most likely a member of the serine protease family. This may function as a virulence protein during pathogenesis by A. solani. The results suggested that the presence of appreciable extracellular proteolytic activity in filamentous fungi may serve as a marker of their phytopathogenicity.

Effect of Chitosan on Rhizome Rot Disease of Turmeric Caused by Pythium aphanidermatum.

Chitosan was evaluated for its potential to induce antifungal hydrolases in susceptible turmeric plant (Curcuma longa L.). Under field conditions, the application of chitosan (crab shell) to turmeric plants by foliar spray method induces defense enzymes such as chitinases and chitosanases. Such an increase in enzyme activity was enhanced by spraying chitosan (0.1% w/v) on leaves of turmeric plants at regular intervals. Gel electrophoresis revealed new chitinase and chitosanase isoforms in leaves of turmeric plants treated with chitosan. Treated turmeric plants showed increased resistance towards rhizome rot disease caused by Pythium aphanidermatum, whereas control plants expressed severe rhizome rot disease. Increased activity of defense enzymes in leaves of chitosan treated turmeric plants may play a role in restricting the development of disease symptoms. The eliciting properties of chitosan make chitosan a potential antifungal agent for the control of rhizome rot disease of turmeric.