Decoding active compounds and molecular targets of herbal medicine by high-throughput metabolomics technology: A systematic review.

Clinical experiences of herbal medicine (HM) have been used to treat a variety of human intractable diseases. As the treatment of diseases using HM is characterized by multi-components and multi-targets, it is difficult to determine the bio-active components, explore the molecular targets and reveal the mechanisms of action. Metabolomics is frequently used to characterize the effect of external disturbances on organisms because of its unique advantages on detecting changes in endogenous small-molecule metabolites. Its systematicity and integrity are consistent with the effective characteristics of HM. After HM intervention, metabolomics can accurately capture and describe the behavior of endogenous metabolites under the disturbance of functional compounds, which will be used to decode the bioactive ingredients of HM and expound the molecular targets. Metabolomics can provide an approach for explaining HM, addressing unclear clinical efficacy and undefined mechanisms of action. In this review, the metabolomics strategy and its applications in HM are systematically introduced, which offers valuable insights for metabolomics methods to characterizing the pharmacological effects and molecular targets of HM.

Exosomes Derived from Human Amniotic Mesenchymal Stem Cells Facilitate Diabetic Wound Healing by Angiogenesis and Enrich Multiple lncRNAs.

BACKGROUND: Diabetic wound healing remains a major challenge due to the impaired functionality of angiogenesis by persistent hyperglycemia. Mesenchymal stem cell exosomes are appropriate candidates for regulating the formation of angiogenesis in tissue repair and regeneration. Here, we explored the effects of exosomes derived from human amniotic mesenchymal stem cell (hAMSC-Exos) on the biological activities of human umbilical vein endothelial cells (HUVECs) treated with high glucose and on diabetic wound healing and investigate lncRNAs related to angiogenesis in hAMSC-Exos. METHODS: hAMSCs and hAMSC-Exos were isolated and identified by flow cytometry or western blot. A series of functional assays such as cell counting kit-8, scratching, transwell and tube formation assays were performed to evaluate the potential effect of hAMSC-Exos on high glucose-treated HUVECs. The effect of hAMSC-Exos on diabetic wound healing were tested by measuring wound closure rates and immunohistochemical staining of CD31. Subsequently, the lncRNAs profiles in hAMSC-Exos and hAMSCs were examined to screen the lncRNAs related to angiogenesis. RESULTS: The isolated hAMSC-Exos had a size range of 30-150 nm and were positive for CD9, CD63 and CD81. The hAMSC-Exos facilitate the functional properties of high glucose-treated HUVECs including the proliferation, migration and the angiogenic activities as well as wound closure and angiogenesis in diabetic wound. hAMSC-Exos were enriched lncRNAs that related to angiogenesis, including PANTR1, H19, OIP5-AS1 and NR2F1-AS1. CONCLUSION: Our findings demonstrated hAMSC-Exos facilitate diabetic wound healing by angiogenesis and contain several exosomal lncRNAs related to angiogenesis, which may represent a promising strategy for diabetic wound healing.

Inhibitory Mechanism of Pinosylvin Monomethyl Ether against Aspergillus flavus.

Control of Aspergillus flavus is beneficial for the agricultural economy and food safety. Stilbenes exhibit antifungal properties through an unknown mechanism. Here, six stilbenes isolated from Cajanus cajan were screened for anti-A. flavus activity. Among them, pinosylvin monomethyl ether (PME) showed the strongest anti-A. flavus activity and has a broad antifungal spectrum with negligible hemolysis within the concentration range measured. PME inhibited the spore germination of A. flavus and the accumulation of aflatoxin B1. Mechanistic studies showed that PME could bind the cell membrane phospholipids, resulting in increased permeability and decreased fluidity. Further metabolic analysis showed that PME caused the lysis of cell membranes and subsequent collapse of spores, which resulted in a cell wall autolysis-like phenotype. Structure-activity relationship analysis revealed the importance of maintaining amphiphilicity harmony by substituent groups for the antifungal activity of stilbenes. Together, natural stilbenes are promising antifungal lead compounds worthy of further exploration and research for potential application in the food, pharmaceutical, and agricultural industries.

Fabrication of chitosan based luminescent nanoprobe with aggregation-induced emission feature through ultrasonic treatment.

Chitosan is an abundant natural polysaccharide that contains a lot of amino and hydroxyl groups. It possesses great potential for biomedical applications owing to its low toxicity, biodegradability and low cost. Herein, a novel chitosan-based fluorescent copolymer (WS-CS-TPA) was designed and synthesized via nucleophilic substitution of hexachlorocyclotriphosphazene (HCCP), water-soluble chitosan (WS-CS) and an aggregation-induced emission (AIE) fluorogen (AIEgen) triphenylamine derivative (TPA-NH2). Under ultrasonic treatment, 1.16 g TPA-NH2 and 1.1 g WS-CS can be conjugated by 0.7 g HCCP at room temperature. The obtained copolymer shows amphiphilic property and could assemble into nanoparticles with size about 100 nm. After self-assembly, TPA-NH2 was aggregated in the core, thus exhibiting superb AIE feature with intense green fluorescence emission in aqueous media. On the other hand, hydrophilic WS-CS was coated on the surface of nanoparticles and endowed their high water dispersibility. Results from preliminary biological assays suggested that WS-CS-TPA can be internalized by cells and exhibits low cytotoxicity, suggesting their great potential for biological imaging and intracellular drug delivery.

Long Non-Coding RNAs profiling in pathogenesis of Verticillium dahliae: New insights in the host-pathogen interaction.

Verticillium dahliae causes vascular wilt disease on cotton (Gossypium hirsutum), resulting in devastating yield loss worldwide. While little is known about the mechanism of long non-coding RNAs (lncRNAs), several lncRNAs have been implicated in numerous physiological processes and diseases. To better understand V. dahliae pathogenesis, lncRNA was conducted in a V. dahliae virulence model. Potential target genes of significantly regulated lncRNAs were predicted using cis/trans-regulatory algorithms. This study provides evidence for lncRNAs' regulatory role in pathogenesis-related genes. Interestingly, lncRNAs were identified and varying in terms of RNA length and nutrient starvation treatments. Efficient pathogen nutrition during the interaction with the host is a requisite factor during infection. Our observations directly link to mutated V. dahliae invasion, explaining infected cotton have lower pathogenicity and lethality compared to V. dahliae. Remarkably, lncRNAs XLOC_006536 and XLOC_000836 involved in the complex regulation of pathogenesis-related genes in V. dahliae were identified. For the first time the regulatory role of lncRNAs in filamentous fungi was uncovered, and it is our contention that elucidation of lncRNAs will advance our understanding in the development and pathogenesis of V. dahliae and offer alternatives in the control of the diseases caused by fungus V. dahliae attack.

Nomogram Predicting Cancer-Specific Death in Parotid Carcinoma: a Competing Risk Analysis.

PURPOSE: Multiple factors have been shown to be tied to the prognosis of individuals with parotid cancer (PC); however, there are limited numbers of reliable as well as straightforward tools available for clinical estimation of individualized mortality. Here, a competing risk nomogram was established to assess the risk of cancer-specific deaths (CSD) in individuals with PC. METHODS: Data of PC patients analyzed in this work were retrieved from the Surveillance, Epidemiology, and End Results (SEER) data repository and the First Affiliated Hospital of Nanchang University (China). Univariate Lasso regression coupled with multivariate Cox assessments were adopted to explore the predictive factors influencing CSD. The cumulative incidence function (CIF) coupled with the Fine-Gray proportional hazards model was employed to determine the risk indicators tied to CSD as per the univariate, as well as multivariate analyses conducted in the R software. Finally, we created and validated a nomogram to forecast the 3- and 5-year CSD likelihood. RESULTS: Overall, 1,467 PC patients were identified from the SEER data repository, with the 3- and 5-year CSD CIF after diagnosis being 21.4% and 24.1%, respectively. The univariate along with the Lasso regression data revealed that nine independent risk factors were tied to CSD in the test dataset (n = 1,035) retrieved from the SEER data repository. Additionally, multivariate data of Fine-Gray proportional subdistribution hazards model illustrated that N stage, Age, T stage, Histologic, M stage, grade, surgery, and radiation were independent risk factors influencing CSD in an individual with PC in the test dataset (p < 0.05). Based on optimization performed using the Bayesian information criterion (BIC), six variables were incorporated in the prognostic nomogram. In the internal SEER data repository verification dataset (n = 432) and the external medical center verification dataset (n = 473), our nomogram was well calibrated and exhibited considerable estimation efficiency. CONCLUSION: The competing risk nomogram presented here can be used for assessing cancer-specific mortality in PC patients.

A Competing Risk Nomogram for Predicting Cancer-Specific Death of Patients With Maxillary Sinus Carcinoma.

OBJECTIVES: Herein, we purposed to establish and verify a competing risk nomogram for estimating the risk of cancer-specific death (CSD) in Maxillary Sinus Carcinoma (MSC) patients. METHODS: The data of individuals with MSC used in this study was abstracted from the (SEER) Surveillance, Epidemiology, and End Results data resource as well as from the First Affiliated Hospital of Nanchang University (China). The risk predictors linked to CSD were identified using the CIF (cumulative incidence function) along with the Fine-Gray proportional hazards model on the basis of univariate analysis coupled with multivariate analysis implemented in the R-software. After that, a nomogram was created and verified to estimate the three- and five-year CSD probability. RESULTS: Overall, 478 individuals with MSC were enrolled from the SEER data resource, with a 3- and 5-year cumulative incidence of CSD after diagnosis of 42.1% and 44.3%, respectively. The Fine-Gray analysis illustrated that age, histological type, N stage, grade, surgery, and T stage were independent predictors linked to CSD in the SEER-training data set (n = 343). These variables were incorporated in the prediction nomogram. The nomogram was well calibrated and it demonstrated a remarkable estimation accuracy in the internal validation data set (n = 135) abstracted from the SEER data resource and the external validation data set (n = 200). The nomograms were well-calibrated and had a good discriminative ability with concordance indexes (c-indexes) of 0.810, 0.761, and 0.755 for the 3- and 5-year prognosis prediction of MSC-specific mortality in the training cohort, internal validation, and external validation cohort, respectively. CONCLUSIONS: The competing risk nomogram constructed herein proved to be an optimal assistant tool for estimating CSD in individuals with MSC.

Synergistic interplay of ABA and BR signal in regulating plant growth and adaptation.

Complex antagonistic interactions between abscisic acid (ABA) and brassinosteroid (BR) signalling pathways have been widely documented. However, whether or how ABA interacts synergistically with BR in plants remains to be elucidated. Here, we report that low, but not high, concentration of ABA increases lamina joint inclination of rice seedling, which requires functional BR biosynthesis and signalling. Transcriptome analyses confirm that about 60% of low-concentration ABA early response genes can be regulated by BR in the same directions. ABA activates BR signal in a fast, limited and short-term manner and the BR-biosynthesis regulatory gene, OsGSR1, plays a key role during this process, whose expression is induced slightly by ABA through transcriptional factor ABI3. Moreover, the early short-term BR signal activation is also important for ABA-mediated salt stress tolerance. Intriguingly, the process and effect of short-term BR signal activation were covered by high concentration of ABA, implying adaptive mechanisms existed in plants to cope with varying degrees of stress.

Molecular evidence for the involvement of cotton GhGLP2, in enhanced resistance to Verticillium and Fusarium Wilts and oxidative stress.

Germin-like proteins (GLPs) are a diverse and ubiquitous family of plant glycoproteins belonging to the cupin super family; they play considerable roles in plant responses against various abiotic and biotic stresses. Here, we provide evidence that GLP2 protein from cotton (Gossypium hirsutum) functions in plant defense responses against Verticillium dahliae, Fusarium oxysporum and oxidative stress. Purified recombinant GhGLP2 exhibits superoxide dismutase (SOD) activity and inhibits spore germination of pathogens. Virus-induced silencing of GhGLP2 in cotton results in increased susceptibility to pathogens, plants exhibited severe wilt on leaves, enhanced vascular browning and suppressed callose deposition. Transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing GhGLP2 showed significant resistance to V. dahliae and F. oxysporum, with reduced mycelia growth, increased callose deposition and cell wall lignification at infection sites on leaves. The enhanced tolerance of GhGLP2-transgenic Arabidopsis to oxidative stress was investigated by methyl viologen and ammonium persulfate treatments, along with increased H2O2 production. Further, the expression of several defense-related genes (PDF1.2, LOX2, and VSP1) or oxidative stress-related genes (RbohD, RbohF) was triggered by GhGLP2. Thus, our results confirmed the involvement of GhGLP2 in plant defense response against Verticillium and Fusarium wilt pathogens and stress conditions.

The cotton GhWIN2 gene activates the cuticle biosynthesis pathway and influences the salicylic and jasmonic acid biosynthesis pathways.

BACKGROUND: Metabolic pathways are interconnected and yet relatively independent. Genes involved in metabolic modules are required for the modules to run. Study of the relationships between genes and metabolic modules improves the understanding of metabolic pathways in plants. The WIN transcription factor activates the cuticle biosynthesis pathway and promotes cuticle biosynthesis. The relationship between the WIN transcription factor and other metabolic pathways is unknown. Our aim was to determine the relationships between the main genes involved in cuticle biosynthesis and those involved in other metabolic pathways. We did this by cloning a cotton WIN gene, GhWIN2, and studying its influence on other pathways. RESULTS: As with other WIN genes, GhWIN2 regulated expression of cuticle biosynthesis-related genes, and promoted cuticle formation. Silencing of GhWIN2 resulted in enhanced resistance to Verticillium dahliae, caused by increased content of salicylic acid (SA). Moreover, silencing of GhWIN2 suppressed expression of jasmonic acid (JA) biosynthesis-related genes and content. GhWIN2 positively regulated the fatty acid biosynthesis pathway upstream of the JA biosynthesis pathway. Silencing of GhWIN2 reduced the content of stearic acid, a JA biosynthesis precursor. CONCLUSIONS: GhWIN2 not only regulated the cuticle biosynthesis pathway, but also positively influenced JA biosynthesis and negatively influenced SA biosynthesis.

GhABP19, a Novel Germin-Like Protein From Gossypium hirsutum, Plays an Important Role in the Regulation of Resistance to Verticillium and Fusarium Wilt Pathogens.

Germin-like proteins (GLPs) are water-soluble plant glycoproteins belonging to the cupin superfamily. The important role of GLPs in plant responses against various abiotic and biotic stresses, especially pathogens, is well validated. However, little is known about cotton GLPs in relation to fungal pathogens. Here, a novel GLP gene was isolated from Gossypium hirsutum and designated as GhABP19. The expression of GhABP19 was upregulated in cotton plants inoculated with Verticillium dahliae and Fusarium oxysporum and in response to treatment with jasmonic acid (JA) but was suppressed in response to salicylic acid treatment. A relatively small transient increase in GhABP19 was seen in H2O2 treated samples. The three-dimensional structure prediction of the GhABP19 protein indicated that the protein has three histidine and one glutamate residues responsible for metal ion binding and superoxide dismutase (SOD) activity. Purified recombinant GhABP19 exhibits SOD activity and could inhibit growth of V. dahliae, F. oxysporum, Rhizoctonia solani, Botrytis cinerea, and Valsa mali in vitro. To further verify the role of GhABP19 in fungal resistance, GhABP19-overexpressing Arabidopsis plants and GhABP19-silenced cotton plants were developed. GhABP19-transgenic Arabidopsis lines showed much stronger resistance to V. dahliae and F. oxysporum infection than control (empty vector) plants did. On the contrary, silencing of GhABP19 in cotton conferred enhanced susceptibility to fungal pathogens, which resulted in necrosis and wilt on leaves and vascular discoloration in GhABP19-silenced cotton plants. The H2O2 content and endogenous SOD activity were affected by GhABP19 expression levels in Arabidopsis and cotton plants after inoculation with V. dahliae and F. oxysporum, respectively. Furthermore, GhABP19 overexpression or silencing resulted in activation or suppression of JA-mediated signaling, respectively. Thus, GhABP19 plays important roles in the regulation of resistance to verticillium and fusarium wilt in plants. These modulatory roles were exerted by its SOD activity and ability to activate the JA pathway. All results suggest that GhABP19 was involved in plant disease resistance.

Enhanced resistance to Verticillium dahliae mediated by an F-box protein GhACIF1 from Gossypium hirsutum.

Avr9/Cf-9-INDUCED F-BOX1 (ACIF1) was first identified during screening of Avr9/Cf-9-elicited genes in tobacco. Further analysis revealed that ACIF1 was required for hypersensitive responses triggered by various elicitors in tobacco and tomato, indicating that it may be involved in various disease resistance. Here, we cloned its cotton (Gossypium hirsutum) homolog GhACIF1, which encodes an F-box protein. We show that GhACIF1 interacts with the putative SKP1-like protein, named GhSKP1. Disease resistance assays show that GhACIF1 enhances resistance to Verticillium dahliae in Arabidopsis plants, while silencing of GhACIF1 confers sensitivity to V. dahliae in cotton. Further analysis show that PevD1 elicitor activates hypersensitive and acquired immune response mediated by GhACIF1. Collectively, these results indicate that GhACIF1 contributes to protection against V. dahliae infection.

GhSNAP33, a t-SNARE Protein From Gossypium hirsutum, Mediates Resistance to Verticillium dahliae Infection and Tolerance to Drought Stress.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate membrane fusion and deliver cargo to specific cellular locations through vesicle trafficking. Synaptosome-associated protein of 25 kDa (SNAP25) is a target membrane SNARE that drives exocytosis by fusing plasma and vesicular membranes. In this study, we isolated GhSNAP33, a gene from cotton (Gossypium hirsutum), encoding a SNAP25-type protein containing glutamine (Q)b- and Qc-SNARE motifs connected by a linker. GhSNAP33 expression was induced by H2O2, salicylic acid, abscisic acid, and polyethylene glycol 6000 treatment and Verticillium dahliae inoculation. Ectopic expression of GhSNAP33 enhanced the tolerance of yeast cells to oxidative and osmotic stresses. Virus-induced gene silencing of GhSNAP33 induced spontaneous cell death and reactive oxygen species accumulation in true leaves at a later stage of cotton development. GhSNAP33-deficient cotton was susceptible to V. dahliae infection, which resulted in severe wilt on leaves, an elevated disease index, enhanced vascular browning and thylose accumulation. Conversely, Arabidopsis plants overexpressing GhSNAP33 showed significant resistance to V. dahliae, with reduced disease index and fungal biomass and elevated expression of PR1 and PR5. Leaves from GhSNAP33-transgenic plants showed increased callose deposition and reduced mycelia growth. Moreover, GhSNAP33 overexpression enhanced drought tolerance in Arabidopsis, accompanied with reduced water loss rate and enhanced expression of DERB2A and RD29A during dehydration. Thus, GhSNAP33 positively mediates plant defense against stress conditions and V. dahliae infection, rendering it a candidate for the generation of stress-resistant engineered cotton.

Mutation of key amino acids in the polygalacturonase-inhibiting proteins CkPGIP1 and GhPGIP1 improves resistance to Verticillium wilt in cotton.

Verticillium wilt, one of the most devastating diseases of cotton (Gossypium hirsutum), causes severe yield and quality losses. Given the effectiveness of plant polygalacturonase-inhibiting proteins (PGIPs) in reducing fungal polygalacturonase (PG) activity, it is necessary to uncover the key functional amino acids to enhance cotton resistance to Verticillium dahliae. To identify novel antifungal proteins, the selectivity of key amino acids was investigated by screening against a panel of relevant PG-binding residues. Based on the obtained results, homologous models of the mutants were established. The docking models showed that hydrogen bonds and structural changes in the convex face in the conserved portion of leucine-rich repeats (LRRs) may be essential for enhanced recognition of PG. Additionally, we successfully constructed Cynanchum komarovii PGIP1 (CkPGIP1) mutants Asp176Val, Pro249Gln, and Asp176Val/Pro249Gln and G. hirsutum PGIP1 (GhPGIP1) mutants Glu169Val, Phe242Gln, and Glu169Val/Phe242Gln with site-directed mutagenesis. The proteins of interest can effectively inhibit VdPG1 activity and V. dahliae mycelial growth in a dose-dependent manner. Importantly, mutants that overproduced PGIP in Arabidopsis and cotton showed enhanced resistance to V. dahliae, with reduced Verticillium-associated chlorosis and wilting. Furthermore, the lignin content was measured in mutant-overexpressing plants, and the results showed enhanced lignification of the xylem, which blocked the spread of V. dahliae. Thus, using site-directed mutagenesis assays, we showed that mutations in CkPGIP1 and GhPGIP1 give rise to PGIP versatility, which allows evolving recognition specificities for PG and is required to promote Verticillium resistance in cotton by restricting the growth of invasive fungal pathogens.

A Cotton Cyclin-Dependent Kinase E Confers Resistance to Verticillium dahliae Mediated by Jasmonate-Responsive Pathway.

Many subunits of the Mediator transcriptional co-activator complex are multifunctional proteins that regulate plant immunity in Arabidopsis. Cotton cyclin-dependent kinase E (GhCDKE), which is a subunit of the cotton (Gossypium hirsutum) Mediator complex, has been annotated, but the biological functions of this gene associated with regulating disease resistance have not been characterized. Here, we cloned GhCDKE from cotton and confirmed that GhCDKE belonged to the E-type CDK family in the phylogenetic tree, and, as in other eukaryotes, we found that GhCDKE interacted with C-type cyclin (GhCycC) by yeast two-hybrid and luciferase complementation imaging assays. Expression of GhCDKE in cotton was induced by Verticillium dahliae infection and MeJA treatment, and silencing of GhCDKE expression in cotton led to enhanced susceptibility to V. dahliae, while overexpression of GhCDKE in Arabidopsis thaliana enhanced resistance to this pathogen. Transgenic expression assay demonstrated that the transcriptional activity of GhPDF1.2pro:LUC in GhCDKE-silenced cotton was dramatically inhibited. In addition, the expression of jasmonic acid (JA)-regulated pathogen-responsive genes was dramatically upregulated in GhCDKE-overexpressed plants after inoculation with V. dahliae, and the roots of GhCDKE-overexpressed A. thaliana were more susceptible to JA treatment. These results indicated that GhCDKE regulates resistance against V. dahliae and that this resistance is mediated by JA response pathway.

A Pectin Methylesterase Inhibitor Enhances Resistance to Verticillium Wilt.

Pectins are major components of the primary plant cell wall, which functions as the primary barrier against pathogens. Pectin methylesterases (PMEs) catalyze the demethylesterification of the homogalacturonan domains of pectin in the plant cell wall. Their activity is regulated by PME inhibitors (PMEIs). Here, we provide evidence that the pectin methylesterase-inhibiting protein GhPMEI3 from cotton (Gossypium hirsutum) functions in plant responses to infection by the fungus Verticillium dahliae GhPMEI3 interacts with PMEs and regulates the expression of a specific fungal polygalacturonase (VdPG1). Ectopic expression of GhPMEI3 increased pectin methyl esterification and limited fungal disease in cotton, while also modulating root elongation. Enzymatic analyses revealed that GhPMEI3 efficiently inhibited the activity of cotton GhPME2/GhPME31. Experiments using transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the GhPMEI3 gene under the control of the CaMV 35S promoter revealed that GhPMEI3 inhibits the endogenous PME activity in vitro. Moreover, the enhanced resistance to V. dahliae was associated with altered VdPG1 expression. Virus-induced silencing of GhPMEI3 resulted in increased susceptibility to V. dahliae Further, we investigated the interaction between GhPMEI3 and GhPME2/GhPME31 using inhibition assays and molecular docking simulations. The peculiar structural features of GhPMEI3 were responsible for the formation of a 1:1 stoichiometric complex with GhPME2/GhPME31. Together, these results suggest that GhPMEI3 enhances resistance to Verticillium wilt. Moreover, GhPMEI3-GhPMEs interactions would be needed before drawing the correlation between structure-function and are crucial for plant development against the ever-evolving fungal pathogens.

Molecular evidence for the involvement of a polygalacturonase-inhibiting protein, GhPGIP1, in enhanced resistance to Verticillium and Fusarium wilts in cotton.

Polygalacturonase-inhibiting protein (PGIP), belonging to a group of plant defence proteins, specifically inhibits endopolygalacturonases secreted by pathogens. Herein, we showed that purified GhPGIP1 is a functional inhibitor of Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum, the two fungal pathogens causing cotton wilt. Transcription of GhPGIP1 was increased in cotton upon infection, wounding, and treatment with defence hormone and H2O2. Resistance by GhPGIP1 was examined by its virus-induced gene silencing in cotton and overexpression in Arabidopsis. GhPGIP1-silenced cotton was highly susceptible to the infections. GhPGIP1 overexpression in transgenic Arabidopsis conferred resistance to the infection, accompanied by enhanced expression of pathogenesis-related proteins (PRs), isochorismate synthase 1 (ICS1), enhanced disease susceptibility 1 (EDS1), and phytoalexin-deficient 4 (PAD4) genes. Transmission electron microscopy revealed cell wall alteration and cell disintegration in plants inoculated with polygalacturonase (PGs), implying its role in damaging the cell wall. Docking studies showed that GhPGIP1 interacted strongly with C-terminal of V. dahliae PG1 (VdPG1) beyond the active site but weakly interacted with C-terminal of F. oxysporum f. sp. vasinfectum (FovPG1). These findings will contribute towards the understanding of the roles of PGIPs and in screening potential combat proteins with novel recognition specificities against evolving pathogenic factors for countering pathogen invasion.

Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That Confers Fungal Resistance in Arabidopsis.

Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species. Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7-66.4% and 56.5-60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.