What type of chromatography was performed on the HiTrap Q HP column?
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Ion-exchange chromatography using a linear NaCl elution gradient from 0 to 1 M.
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What type of chromatography was performed on the HiTrap Q HP column?
Ion-exchange chromatography using a linear NaCl elution gradient from 0 to 1 M.
How were the total RNA concentrations adjusted in the experiment?
They were adjusted to be the same for each sample.
What was the effect of PevD1 on fluorescence in cotton cells?
Strong cell fluorescence was observed 20 minutes after the addition of PevD1.
What is the role of the elicitor PevD1 in cotton?
PevD1 triggers innate immunity in cotton, playing an important role in controlling cotton wilt disease.
What method was used to detect H2O2 production in cell culture?
Chemiluminescence assay.
How was lignin content evaluated in cotton stems?
By extracting and measuring the absorbance of TGA derivatives.
What is the effect of PevD1 on TMV lesions in tobacco plants?
It reduces the number and size of TMV lesions.
What was the temperature and duration for incubating the cell sample with PevD1?
1 hour at 28 °C.
What was detected to research the resistance mechanisms of PevD1-treated cotton seedlings?
The expression levels of cotton defense-related genes from 0 to 72 hours after inoculation with V. dahliae.
What role does cadinene synthase play in cotton?
It is involved in the biosynthetic pathway of a terpenoid that is a significant phytoalexin.
What is the role of oligosaccharides from B. cinerea in plant defense?
They induce typical PTI responses, such as oxidative burst, cell wall lignification, and resistance to B. cinerea.
How does PevD1 treatment affect the activities of defense enzymes?
PevD1 treatment significantly increases the activities of PAL, POD, and PPO, enhancing resistance against wilt pathogens.
What was used as a protein standard in the BCA Protein Assay?
Bovine serum albumin (1.0 mg ml - 1).
What method was used to observe fluorescence in cotton suspension cells?
An Olympus laser confocal scanning microscope.
What was the photoperiod for maintaining the cotton plants?
16 hours light and 8 hours dark.
How did lignin accumulation change after PevD1 treatment?
Lignin deposition increased about twofold compared to the control at 96 hours after treatment.
What are the mechanisms of resistance conferred by PevD1?
They involve the production of secondary metabolites, lignin deposition, and cell wall modifications.
What was the maximum disease reduction achieved with PevD1 treatment?
38.16% on the 15th day post V. dahliae inoculation.
When did PAL activity peak after PevD1 treatment?
At 96 hours.
Which pathogenesis-related genes are enhanced by PevD1?
b-1,3-glucanase, chitinase, and cadinene synthase.
When did the expression levels of acidic chitinase genes peak after PevD1 treatment?
At 24 hours.
What do some fungal effectors or elicitors trigger in plants?
Broad-spectrum defense responses, including cell wall reinforcement and biosynthesis of antimicrobial compounds.
What is the significance of lignin deposition in cotton plants?
Lignin deposition reinforces cell walls, creating barriers to limit pathogen infection.
What method was used to analyze the expression of PR genes induced by PevD1?
Semi-quantitative RT-PCR.
What materials were found in the cells of cotton plants in response to V. dahliae infection?
Granular materials and amorphous material were present in the cells.
What do successful pathogens secrete to interfere with plant defense?
Effectors.
At what concentration of PevD1 was the highest disease reduction observed?
16 µg/ml.
Which enzymes involved in lignin synthesis were detected after PevD1 treatment?
PAL, PPO, and POD.
What common defense responses are generated in plants during early stages of pathogen interaction?
Generation of reactive oxygen species (ROS) and nitric oxide (NO).
What was the concentration of PevD1 used for treating cotton leaves?
8 µg/ml.
What are the limitations of traditional methods for controlling Verticillium wilt in cotton?
Traditional breeding has not produced resistant cultivars, and effective chemical pesticides are scarce.
What are the core genes of the phenylpropanoid pathway in cotton?
PAL, C4H1, and 4CL are the core genes involved in the phenylpropanoid pathway.
What medium was used to culture Verticillium dahliae?
Potato dextrose agar medium.
What is PevD1 and its significance in cotton?
PevD1 is a protein that induces resistance against the cotton wilt pathogen V. dahliae.
What role does lignin play in plant responses to V. dahliae?
Lignin acts as a physical barrier that enhances cell wall resistance to mechanical pressure during fungal penetration.
What method was used to monitor nitric oxide accumulation in leaves?
Using the NO-sensitive dye DAF-FM DA.
At what concentration was recombinant PevD1 effective in improving cotton resistance?
As low as 4 µg/ml.
How much did PPO and POD activities increase after PevD1 treatment?
They were three to fourfold higher than the control at their peak.
What is the molecular weight of the PevD1 protein?
16.23 kDa.
What are the two main types of fungal effector proteins?
Extracellular effectors and cytoplasmic effectors.
What is the role of PAL in the phenylpropanoid pathway?
PAL is responsible for the synthesis of defense-related compounds, creating a physical barrier against pathogens.
What types of pathogens are plants exposed to in nature?
Viruses, bacteria, oomycetes, and fungi.
What was the purpose of using the cotton ubiquitin gene in the study?
It was used as a control in the PCR analysis.
How was the degree of wilt disease assessed?
Divided into five grades based on the percentage of affected leaf area.
What potential application does PevD1 have in agriculture?
It is expected to be a novel biopesticide for controlling cotton Verticillium wilt disease.
How long after elicitor treatment did ROS production reach its maximum?
Approximately 4 minutes.
Why is Verticillium wilt a major threat to cotton production?
It is a soil-borne vascular disease caused by Verticillium dahliae, particularly destructive in warmer regions.
Which key genes are part of the core phenylpropanoid pathway in cotton?
PAL, cinnamate 4-hydroxylase (C4H1), and 4-coumarate: CoA ligase (4CL).
What was measured to assess the effects of PevD1 treatment on leaves?
The activities of PAL, PPO, and POD.
What role do elicitors play in host defense against pathogens?
Elicitors increase lignin synthesis and deposition to prevent pathogen infections in cotton seedlings.
How were the cotton seeds sterilized before planting?
Immersed in 15% H2O2 for 1–3 hours and rinsed with sterile water.
How was hydrogen peroxide (H2O2) production detected in cotton leaves?
Using 3,3'-diaminobenzidine (DAB)-HCl and observing under a microscope.
What was used as a control when treating cotton leaves with PevD1?
Water was used as a control.
What pathogen does PevD1 enhance resistance against in cotton?
Verticillium dahliae.
What is PevD1 and its significance?
PevD1 is a protein elicitor from V. dahliae that induces systemic acquired resistance in tobacco plants.
Which enzymes' activities were measured in the leaves after PevD1 treatment?
Peroxidase (POD), phenylalanine ammonia-lyase (PAL), and polyphenol oxidase (PPO).
What is systemic acquired resistance (SAR)?
A process where defense reactions extend from the local site of infection to non-infected cells.
When did the PevD1-induced cadinene synthase gene expression reach its highest point?
At 24–48 hours post-inoculation.
What is PevD1 and its significance in plant immunity?
PevD1 is a small secreted protein from V. dahliae that triggers resistance in tobacco and induces PR gene expression in cotton.
What physiological changes occur in cotton plants after PevD1 treatment?
Increased expression of PR genes and core genes in the lignin metabolism pathway, enhancing disease resistance.
What effect does PevD1 leaf infiltration have on cotton plants?
It significantly increases lignin deposition and the formation of incrassated walls in xylem vessels.
What is the first line of defense in plants against pathogens?
Pattern-triggered immunity (PTI) activated by the recognition of MAMPs/PAMPs.
What was the relative molecular weight of the expressed PevD1 protein?
17 kDa.
What effect did PevD1 have on cotton seedlings infected by V. dahliae?
It promoted disease resistance.
What changes were observed in vessel walls after PevD1 treatment?
Vessel walls were thickened and reinforced compared to the control after 3 and 5 days.
What was the purpose of embedding stem samples in the experiment?
To observe vessels and stem cell structures after PevD1 treatment.
What statistical method was used to analyze the data from the experiments?
Tukey’s HSD test.
What is the significance of the phenylpropanoid metabolism pathway in cotton defense?
It includes key genes like PAL, C4H1, and 4CL that are upregulated by PevD1.
When were the expression levels of core phenylpropanoid pathway genes increased after PevD1 treatment?
At 8 hours.
What effect does PevD1 have on resistance-related gene transcripts in cotton?
PevD1 induces moderate accumulation of transcripts for genes like β-1,3-glucanase and chitinases.
What strain of fungus was isolated from diseased cotton?
Verticillium dahliae strain HX-8.
What is the purpose of using DAF-FM DA in the experiment?
To measure NO production in suspension cells.
How does lignin deposition contribute to plant defense?
It forms a physical barrier to fungal penetration and expansion.
What was the purpose of the PevD1 treatment in the bioassay?
To control disease symptoms after pathogen inoculation.
What is the second line of defense in plants called?
Effector-triggered immunity (ETI).
What is the role of elicitors in plant resistance?
Some elicitors improve plant resistance by triggering defense responses.
What early events were induced in cotton by the PevD1 elicitor?
Accumulation of H2O2 and an ROS burst.
What types of responses does PevD1 induce in cotton?
Hydrogen peroxide production, nitric oxide generation, lignin deposition, and defense enzyme activation.
What was the observed effect of PevD1 on ROS and NO release in cotton?
ROS burst was detected after PevD1 treatment, while NO release was also affected.
Which genes showed enhanced expression at 12 hours after PevD1 treatment?
b-1,3-glucanase and basic chitinase genes.
How do early signaling molecules like NO and ROS contribute to plant defense?
They are involved in the defense response and trigger signaling pathways that regulate physiological responses through gene expression.
