Bacillus thuringiensis (Bt) cotton production is challenged by two main problems, i.e., the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs.  Therefore, increasing the Bt protein concentration at the boll stage, especially in bolls, has become the main goal for increasing insect resistance in cotton.  In this study, two protein degradation inhibitors (ethylene diamine tetra acetic acid (EDTA) and leupeptin) were sprayed on the bolls, subtending leaves, and whole cotton plants at the peak flowering stage of two Bt cultivars (medium maturation Sikang 1 (SK1) and early maturation Zhongmian 425 (ZM425) in 2019 and 2020.  The Bt protein content and protein degradation metabolism were assessed.  The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8% and 25.0 to 38.6% in the treated bolls of SK1 and ZM425 respectively, while they were decreased in the subtending leaves of these treated bolls.  In the treated leaves, the Bt protein concentrations increased by 7.6 to 23.5% and 11.2 to 14.9% in SK1 and ZM425, respectively.  The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves.  The Bt protein concentrations in bolls were higher, increasing by 22.5 to 31.0% and 19.6 to 32.5% for SK1 and ZM425, respectively.  The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents, protease and peptidase activities and significant enhancements in soluble protein contents.  These results indicated that inhibiting protein degradation could improve the protein content, thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants.  Therefore, the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

The effects of mepiquat chloride (DPC) on the Cry1Ac protein content in Bacillus thuringiensis (Bt) cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and -efficiency cotton cultivation.  This study was conducted using Bt cotton cultivar ‘Sikang 3’ during the 2020 and 2021 growing seasons at Yangzhou University Farm, Yangzhou, Jiangsu Province, China.  Potted cotton plants were exposed to high temperature and drought stress, and sprayed with either 20 mg L⁻¹ DPC or water (CK).  Seven days after treatment, the Cry1Ac protein content, α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content were measured, and transcriptome sequencing was performed.  DESeq was used for differential gene analysis.  Under the DPC treatment, the Cry1Ac protein content increased by 4.7–11.9% compared to CK.  The α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content all increased.  Transcriptome analysis revealed 7,542 upregulated genes and 10,449 downregulated genes for DPC vs. CK.  Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses showed that the differentially expressed genes were mainly involved in biological processes, such as carbon and amino acid metabolism.  For example, genes encoding 6-phosphofructokinase, pyruvate kinase, glutamic pyruvate transaminase, pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, glutamate synthase, 1-pyrroline-5-carboxylate dehydrogenase, glutamic oxaloacetic transaminase, amino-acid N-acetyltransferase, and acetylornithine deacetylase were all significantly upregulated.  The DPC treatment increased pyruvate, α-ketoglutarate, and oxaloacetate by increasing the operational rate of the glycolytic pathway of the citric acid cycle.  It also significantly upregulated the genes encoding glutamate synthase, pyrrolidine-5-carboxylic acid dehydrogenase, glutamate oxaloacetate transaminase, and N-acetylglutamate synthetase, while it downregulated the genes encoding glutamine synthetase.  Therefore, the synthesis of aspartic acid, glutamic acid, pyruvate, and arginine increased after treatment with DPC, and the Cry1Ac protein content was increased by regulating carbon and amino acid metabolism.

Nicotinamide mononucleotide (NMN), a precursor in nicotinamide adenine dinucleotide (NAD) biosynthesis, has long been recognized for its pivotal role in medicine. Recent investigations have suggested its potential as a plant immunity inducer for controlling fungal diseases. However, whether NMN confers plant broad-spectrum resistance against diverse phytopathogens, and its underlying mechanisms remain ambiguous. In this study, we investigate the effect of NMN against multiple phytopathogens in tobacco. Our results demonstrate that tobacco pretreated with NMN exhibits enhanced resistance against Rastonia solanacearum CQPS-1, Pseudomonas syringae DC3000 ∆hopQ1-1, Phytophthora parasitica, and tobacco mosaic virus (TMV). NMN displays effectiveness within the concentration range of 50-600 μM, with 75 μM NMN exhibiting the most pronounced effect. The impact of NMN pretreatment could persist for up to 10 days. Beyond tobacco, NMN pretreatment enhances disease resistance in tomato and pepper plants against diverse pathogens, underscoring NMN’s capacity to confer broad-spectrum disease resistance in crops. Moreover, RT-qPCR analysis reveals that NMN significantly upregulates the expression of the pattern-triggered immunity (PTI) marker gene NbCYP71D20 and salicylic acid (SA) marker gene NbPR1a. This suggests that NMN enhances plant resistance by inducing both PTI and SA-mediated immunity. Interestingly, the positive impact of NMN on plant disease resistance is not significantly compromised in both NMN adenylyltransferase (NMNAT)-silenced plants and NAD receptor mutant lecrk-I.8, suggesting the existence of NAD-independent signaling pathways for NMN-induced plant immunity. In conclusion, our study establishes that the bioactive molecule NMN imparts broad-spectrum disease resistance in plants, offering a simple, environmental-friendly, and promising strategy for safeguarding crops against diverse phytopathogens. These findings also provide valuable insights for future in-depth studies into the functional mechanisms of NMN. 

Simultaneously enhancing plant growth and disease resistance is an ideal goal in Agriculture. Significant efforts have been made to promote plant growth or immunity through the use of biological reagents, such as the application of beneficial microbes and plant immunity inducers. However, balancing plant immunity and growth remains a challenging task. In this study, we engineered the plant growth-promoting bacterium Bacillus subtilis OKB105 to express a secreted microbial pattern, flg22, and accessed its activity in enhancing both plant growth and disease resistance. The OKB105 (flg22) strain exhibited plant growth-promoting activity similar to the OKB105 strain containing an empty vector, OKB105 (EV). Furthermore, the OKB105 (flg22) strain significantly enhanced plant resistance against two distinct pathogens, Pseudomonas syringae DC3000 ΔhopQ1-1 and Phytophthora parasitica, compared to OKB105 (EV), confirming that the engineered OKB105 (flg22) effectively enhances plant disease resistance. Interestingly, root irrigation with OKB105 (flg22) also markedly boosted the plant’s aboveground resistance to pathogens compared to OKB105 (EV). We further demonstrated that OKB105 (flg22) can be applied to confer resistance to pathogens in other plants that recognize flg22. Finally, RNA-Seq and qRT-PCR analyses illustrated that OKB105 (flg22) effectively induced the expression of defense-related genes in pattern-triggered immunity. Our results prove that employing an engineered beneficial microbe expressing a microbial pattern is a promising strategy for simultaneously enhancing plant growth and immunity. 

Pepper fruit is highly favored for its spicy taste, diverse flavors, and high nutritional value. The proper development of its flower and fruit directly determines the quality of pepper fruit. The YABBY gene family exhibits diverse functions in growth and development, which is crucial to the identity of plant flower organs, but its specific role in pepper is still unclear. In this study, nine CaYABBY genes were identified and characterized in pepper. Most CaYABBY genes were highly expressed in reproductive organs, albeit with varying patterns of expression. The CaYABBY5 gene, uniquely expressed in petals and carpels, has been demonstrated to modulate floral organ determinacy and fruit shape through gene silencing in pepper and ectopic expression in tomato. Protein interaction analysis revealed an interacting protein SEPALLATA3-like protein (SEP3), exhibiting a similar expression profile to that of CaYABBY5. These findings suggest that CaYABBY5 may modulate the morphogenesis of floral organs and fruits by interacting with CaSEP3. This study provided valuable insights into the classification and function of CaYABBY genes in pepper.