International Journal of Molecular Sciences

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21 pages, 3451 KiB

Open AccessArticle

A New Approach for CRISPR/Cas9 Editing and Selection of Pathogen-Resistant Plant Cells of Wine Grape cv. ‘Merlot’

by Anastasia Fizikova, Zhanneta Tukhuzheva, Lada Zhokhova, Varvara Tvorogova and Ludmila Lutova

Int. J. Mol. Sci. 2024, 25(18), 10011; https://doi.org/10.3390/ijms251810011 - 17 Sep 2024

Cited by 1 | Viewed by 1413

Abstract

Grape is one of the most economically significant berry crops. Owing to the biological characteristics of grapes, such as the long juvenile period (5–8 years), high degree of genome heterozygosity, and the frequent occurrence of inbreeding depression, homozygosity during crossbreeding leads to loss [...] Read more.

Grape is one of the most economically significant berry crops. Owing to the biological characteristics of grapes, such as the long juvenile period (5–8 years), high degree of genome heterozygosity, and the frequent occurrence of inbreeding depression, homozygosity during crossbreeding leads to loss of varietal characteristics and viability. CRISPR/Cas editing has become the tool of choice for improving elite technical grape varieties. This study provides the first evidence of a decrease in the total fraction of phenolic compounds and an increase in the concentration of peroxide compounds in grape callus cells upon the addition of chitosan to the culture medium. These previously unreported metabolic features of the grape response to chitosan have been described and used for the first time to increase the probability of selecting plant cells with MLO7 knockout characterised by an oxidative burst in response to the presence of a pathogen modulated by chitosan in the high-metabolite black grape variety ‘Merlot’. This was achieved by using a CRISPR/Cas9 editing vector construction with the peroxide sensor HyPer as a reporter. This research represents the first CRISPR/Cas9 editing of ‘Merlot’, one of the most economically important elite technical grape varieties. Full article

(This article belongs to the Special Issue Plant Molecular Breeding and Resistance: Phenomics, Genomics and Gene Editing)

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10 pages, 256 KiB

Open AccessCommunication

Investigation of 2,4-Dihydroxylaryl-Substituted Heterocycles as Inhibitors of the Growth and Development of Biotrophic Fungal Pathogens Associated with the Most Common Cereal Diseases

by Klaudia Rząd, Aleksandra Nucia, Weronika Grzelak, Joanna Matysiak, Krzysztof Kowalczyk, Sylwia Okoń and Arkadiusz Matwijczuk

Int. J. Mol. Sci. 2024, 25(15), 8262; https://doi.org/10.3390/ijms25158262 - 29 Jul 2024

Viewed by 635

Abstract

Climate change forces agriculture to face the rapidly growing virulence of biotrophic fungal pathogens, which in turn drives researchers to seek new ways of combatting or limiting the spread of diseases caused by the same. While the use of agrochemicals may be the [...] Read more.

Climate change forces agriculture to face the rapidly growing virulence of biotrophic fungal pathogens, which in turn drives researchers to seek new ways of combatting or limiting the spread of diseases caused by the same. While the use of agrochemicals may be the most efficient strategy in this context, it is important to ensure that such chemicals are safe for the natural environment. Heterocyclic compounds have enormous biological potential. A series of heterocyclic scaffolds (1,3,4-thiadiazole, 1,3-thiazole, 1,2,4-triazole, benzothiazine, benzothiadiazine, and quinazoline) containing 2,4-dihydroxylaryl substituents were investigated for their ability to inhibit the growth and development of biotrophic fungal pathogens associated with several important cereal diseases. Of the 33 analysed compounds, 3 were identified as having high inhibitory potential against Blumeria and Puccinia fungi. The conducted research indicated that the analysed compounds can be used to reduce the incidence of fungal diseases in cereals; however, further thorough research is required to investigate their effects on plant–pathogen systems, including molecular studies to determine the exact mechanism of their activity. Full article

(This article belongs to the Special Issue Plant Molecular Breeding and Resistance: Phenomics, Genomics and Gene Editing)

15 pages, 4763 KiB

Open AccessArticle

PpGATA21 Enhances the Expression of PpGA2ox7 to Regulate the Mechanism of Cerasus humilis Rootstock-Mediated Dwarf in Peach Trees

by Xiuzhen Li, Ruxin Wang, Yuman Wang, Xueqiang Li, Qiaofang Shi and Yihe Yu

Int. J. Mol. Sci. 2024, 25(13), 7402; https://doi.org/10.3390/ijms25137402 - 5 Jul 2024

Viewed by 764

Abstract

Dwarfing rootstocks enhance planting density, lower tree height, and reduce both labor in peach production. Cerasus humilis is distinguished by its dwarf stature, rapid growth, and robust fruiting capabilities, presenting substantial potential for further development. In this study, Ruipan 4 was used as [...] Read more.

Dwarfing rootstocks enhance planting density, lower tree height, and reduce both labor in peach production. Cerasus humilis is distinguished by its dwarf stature, rapid growth, and robust fruiting capabilities, presenting substantial potential for further development. In this study, Ruipan 4 was used as the scion and grafted onto Amygdalus persica and Cerasus humilis, respectively. The results indicate that compared to grafting combination R/M (Ruipan 4/Amygdalus persica), grafting combination R/O (Ruipan 4/Cerasus humilis) plants show a significant reduction in height and a significant increase in flower buds. RNA-seq indicates that genes related to gibberellin (GA) and auxin metabolism are involved in the dwarfing process of scions mediated by C. humilis. The expression levels of the GA metabolism-related gene PpGA2ox7 significantly increased in R/O and are strongly correlated with plant height, branch length, and internode length. Furthermore, GA levels were significantly reduced in R/O. The transcription factor PpGATA21 was identified through yeast one-hybrid screening of the PpGA2ox7 promoter. Yeast one-hybrid (Y1H) and dual-luciferase reporter (DLR) demonstrate that PpGATA21 can bind to the promoter of PpGA2ox7 and activate its expression. Overall, PpGATA21 activates the expression of the GA-related gene PpGA2ox7, resulting in reduced GA levels and consequent dwarfing of plants mediated by C. humilis. This study provides new insights into the mechanisms of C. humilis and offers a scientific foundation for the dwarfing and high-density cultivation of peach trees. Full article

(This article belongs to the Special Issue Plant Molecular Breeding and Resistance: Phenomics, Genomics and Gene Editing)

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23 pages, 7427 KiB

Open AccessArticle

Physiological and Proteome Analysis of the Effects of Chitosan Oligosaccharides on Salt Tolerance of Rice Seedlings

by Xiangyu Qian, Yaqing He, Lu Zhang, Xianzhen Li and Wenzhu Tang

Int. J. Mol. Sci. 2024, 25(11), 5953; https://doi.org/10.3390/ijms25115953 - 29 May 2024

Cited by 1 | Viewed by 980

Abstract

Rice (Oryza sativa L.) is an important social-economic crop, and rice seedlings are easily affected by salt stress. Chitosan oligosaccharide (COS) plays a positive role in promoting plant growth and development. To gain a better understanding of the salt tolerance mechanism of [...] Read more.

Rice (Oryza sativa L.) is an important social-economic crop, and rice seedlings are easily affected by salt stress. Chitosan oligosaccharide (COS) plays a positive role in promoting plant growth and development. To gain a better understanding of the salt tolerance mechanism of rice under the action of COS, Nipponbare rice seedlings were selected as the experimental materials, and the physiological and biochemical indexes of rice seedlings in three stages (normal growth, salt stress and recovery) were measured. Unlabelled quantitative proteomics technology was used to study differential protein and signaling pathways of rice seedlings under salt stress, and the mechanism of COS to improve rice tolerance to salt stress was elucidated. Results showed that after treatment with COS, the chlorophyll content of rice seedlings was 1.26 times higher than that of the blank group (CK). The root activity during the recovery stage was 1.46 times that of the CK group. The soluble sugar in root, stem and leaf increased by 53.42%, 77.10% and 9.37%, respectively. The total amino acid content increased by 77% during the stem recovery stage. Furthermore, the malondialdehyde content in root, stem and leaf increased by 21.28%, 26.67% and 32.69%, respectively. The activity of oxide dismutase (SOD), peroxidase (POD) and oxygenase (CAT) were increased. There were more differentially expressed proteins in the three parts of the experimental group than in the CK group. Gene Ontology (GO) annotation of these differentially expressed proteins revealed that the experimental group was enriched for more entries. Then, through the Kyoto Encyclopedia of Genes and Genomes (KEGG), the top ten pathways enriched with differentially expressed proteins in the two groups (COS and CK groups) were utilized, and a detailed interpretation of the glycolysis and photosynthesis pathways was provided. Five key proteins, including phosphofructokinase, fructose bisphosphate aldolases, glycer-aldehyde-3-phosphate dehydrogenase, enolase and pyruvate kinase, were identified in the glycolysis pathway. In the photosynthesis pathway, oxygen evolution enhancement proteins, iron redox proteins and ferredoxin-NADPH reductase were the key proteins. The addition of COS led to an increase in the abundance of proteins, a response of rice seedlings to salt stress. COS helped rice seedlings resist salt stress. Furthermore, using COS as biopesticides and biofertilizers can effectively increase the utilization of saline-affected farmland, thereby contributing to the alleviating of the global food crisis. Full article

(This article belongs to the Special Issue Plant Molecular Breeding and Resistance: Phenomics, Genomics and Gene Editing)

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22 pages, 5854 KiB

Open AccessArticle

Revealing the Complete Bispecific Phosphatase Genes (DUSPs) across the Genome and Investigating the Expression Patterns of GH_A11G3500 Resistance against Verticillium wilt

by Yahui Deng, Xiaojuan Deng, Jieyin Zhao, Shuo Ning, Aixing Gu, Quanjia Chen and Yanying Qu

Int. J. Mol. Sci. 2024, 25(8), 4500; https://doi.org/10.3390/ijms25084500 - 19 Apr 2024

Viewed by 959

Abstract

DUSPs, a diverse group of protein phosphatases, play a pivotal role in orchestrating cellular growth and development through intricate signaling pathways. Notably, they actively participate in the MAPK pathway, which governs crucial aspects of plant physiology, including growth regulation, disease resistance, pest resistance, [...] Read more.

DUSPs, a diverse group of protein phosphatases, play a pivotal role in orchestrating cellular growth and development through intricate signaling pathways. Notably, they actively participate in the MAPK pathway, which governs crucial aspects of plant physiology, including growth regulation, disease resistance, pest resistance, and stress response. DUSP is a key enzyme, and it is the enzyme that limits the rate of cell metabolism. At present, complete understanding of the DUSP gene family in cotton and its specific roles in resistance to Verticillium wilt (VW) remains elusive. To address this knowledge gap, we conducted a comprehensive identification and analysis of four key cotton species: Gossypium arboreum, Gossypium barbadense, Gossypium hirsutum, and Gossypium raimondii. The results revealed the identification of a total of 120 DUSP genes in the four cotton varieties, which were categorized into six subgroups and randomly distributed at both ends of 26 chromosomes, predominantly localized within the nucleus. Our analysis demonstrated that closely related DUSP genes exhibited similarities in terms of the conserved motif composition and gene structure. A promoter analysis performed on the GhDUSP gene promoter revealed the presence of several cis-acting elements, which are associated with abiotic and biotic stress responses, as well as hormone signaling. A tissue expression pattern analysis demonstrated significant variations in GhDUSP gene expression under different stress conditions, with roots exhibiting the highest levels, followed by stems and leaves. In terms of tissue-specific detection, petals, leaves, stems, stamens, and receptacles exhibited higher expression levels of the GhDUSP gene. The gene expression analysis results for GhDUSPs under stress suggest that DUSP genes may have a crucial role in the cotton response to stress in cotton. Through Virus-Induced Gene Silencing (VIGS) experiments, the silencing of the target gene significantly reduced the resistance efficiency of disease-resistant varieties against Verticillium wilt (VW). Consequently, we conclude that GH_A11G3500-mediated bispecific phosphorylated genes may serve as key regulators in the resistance of G. hirsutum to Verticillium wilt (VW). This study presents a comprehensive structure designed to provide an in-depth understanding of the potential biological functions of cotton, providing a strong foundation for further research into molecular breeding and resistance to plant pathogens. Full article

(This article belongs to the Special Issue Plant Molecular Breeding and Resistance: Phenomics, Genomics and Gene Editing)

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13 pages, 1965 KiB

Open AccessArticle

Patch Track Software for Measuring Kinematic Phenotypes of Arabidopsis Roots Demonstrated on Auxin Transport Mutants

by Ashley R. Henry, Nathan D. Miller and Edgar P. Spalding

Int. J. Mol. Sci. 2023, 24(22), 16475; https://doi.org/10.3390/ijms242216475 - 18 Nov 2023

Cited by 1 | Viewed by 1244

Abstract

Plant roots elongate when cells produced in the apical meristem enter a transient period of rapid expansion. To measure the dynamic process of root cell expansion in the elongation zone, we captured digital images of growing Arabidopsis roots with horizontal microscopes and analyzed [...] Read more.

Plant roots elongate when cells produced in the apical meristem enter a transient period of rapid expansion. To measure the dynamic process of root cell expansion in the elongation zone, we captured digital images of growing Arabidopsis roots with horizontal microscopes and analyzed them with a custom image analysis program (PatchTrack) designed to track the growth-driven displacement of many closely spaced image patches. Fitting a flexible logistics equation to patch velocities plotted versus position along the root axis produced the length of the elongation zone (mm), peak relative elemental growth rate (% h⁻¹), the axial position of the peak (mm from the tip), and average root elongation rate (mm h⁻¹). For a wild-type root, the average values of these kinematic traits were 0.52 mm, 23.7% h⁻¹, 0.35 mm, and 0.1 mm h⁻¹, respectively. We used the platform to determine the kinematic phenotypes of auxin transport mutants. The results support a model in which the PIN2 auxin transporter creates an area of expansion-suppressing, supraoptimal auxin concentration that ends 0.1 mm from the quiescent center (QC), and that ABCB4 and ABCB19 auxin transporters maintain expansion-limiting suboptimal auxin levels beginning approximately 0.5 mm from the QC. This study shows that PatchTrack can quantify dynamic root phenotypes in kinematic terms. Full article

(This article belongs to the Special Issue Plant Molecular Breeding and Resistance: Phenomics, Genomics and Gene Editing)

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16 pages, 3255 KiB

Open AccessArticle

Resistant and Susceptible Pinus thunbergii ParL. Show Highly Divergent Patterns of Differentially Expressed Genes during the Process of Infection by Bursaphelenchus xylophilus

by Tingyu Sun, Mati Ur Rahman, Xiaoqin Wu and Jianren Ye

Int. J. Mol. Sci. 2023, 24(18), 14376; https://doi.org/10.3390/ijms241814376 - 21 Sep 2023

Cited by 2 | Viewed by 1341

Abstract

Pine wilt disease (PWD) is a devastating disease that threatens pine forests worldwide, and breeding resistant pines is an important management strategy used to reduce its impact. A batch of resistant seeds of P. thunbergii was introduced from Japan. Based on the resistant [...] Read more.

Pine wilt disease (PWD) is a devastating disease that threatens pine forests worldwide, and breeding resistant pines is an important management strategy used to reduce its impact. A batch of resistant seeds of P. thunbergii was introduced from Japan. Based on the resistant materials, we obtained somatic plants through somatic embryogenesis. In this study, we performed transcriptome analysis to further understand the defense response of resistant somatic plants of P. thunbergii to PWD. The results showed that, after pine wood nematode (PWN) infection, resistant P. thunbergii stimulated more differential expression genes (DEGs) and involved more regulatory pathways than did susceptible P. thunbergii. For the first time, the alpha-linolenic acid metabolism and linoleic acid metabolism were intensively observed in pines resisting PWN infection. The related genes disease resistance protein RPS2 (SUMM2) and pathogenesis-related genes (PR1), as well as reactive oxygen species (ROS)-related genes were significantly up-expressed in order to contribute to protection against PWN inoculation in P. thunbergii. In addition, the diterpenoid biosynthesis pathway was significantly enriched only in resistant P. thunbergii. These findings provided valuable genetic information for future breeding of resistant conifers, and could contribute to the development of new diagnostic tools for early screening of resistant pine seedlings based on specific PWN-tolerance-related markers. Full article

(This article belongs to the Special Issue Plant Molecular Breeding and Resistance: Phenomics, Genomics and Gene Editing)

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Review

Jump to: Research

13 pages, 1147 KiB

Open AccessReview

CRISPR Variants for Gene Editing in Plants: Biosafety Risks and Future Directions

by Ali Movahedi, Soheila Aghaei-Dargiri, Hongyan Li, Qiang Zhuge and Weibo Sun

Int. J. Mol. Sci. 2023, 24(22), 16241; https://doi.org/10.3390/ijms242216241 - 13 Nov 2023

Cited by 3 | Viewed by 4947

Abstract

The CRISPR genome editing technology is a crucial tool for enabling revolutionary advancements in plant genetic improvement. This review shows the latest developments in CRISPR/Cas9 genome editing system variants, discussing their benefits and limitations for plant improvement. While this technology presents immense opportunities [...] Read more.

The CRISPR genome editing technology is a crucial tool for enabling revolutionary advancements in plant genetic improvement. This review shows the latest developments in CRISPR/Cas9 genome editing system variants, discussing their benefits and limitations for plant improvement. While this technology presents immense opportunities for plant breeding, it also raises serious biosafety concerns that require careful consideration, including potential off-target effects and the unintended transfer of modified genes to other organisms. This paper highlights strategies to mitigate biosafety risks and explores innovative plant gene editing detection methods. Our review investigates the international biosafety guidelines for gene-edited crops, analyzing their broad implications for agricultural and biotechnology research and advancement. We hope to provide illuminating and refined perspectives for industry practitioners and policymakers by evaluating CRISPR genome enhancement in plants. Full article

(This article belongs to the Special Issue Plant Molecular Breeding and Resistance: Phenomics, Genomics and Gene Editing)

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