Part:BBa_K4628008:Experience

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General use

Transforming the ID sequence of the original Pikm-1 to an Enhancer nanoantibody targeting GFP, thereby artificially altering its immune inducible stimuli.

Phenotype observation

Tobacco was irradiated with 488 nm excitation light to observe the removal of GFP fluorescence

It can be seen that compared with the positive control, the negative control has a clear fluorescence clearance.

Figure 1: Phenotypic observations after transient tobacco

Immune clearance

Next, we will quantify the clearance of GFP (Total protein fluorescence intensity & WB) at the molecular level and confirm that it is cleared by plant’s immune system (ROS detection)

Protein Fluorescent Analysis

After one week of injection, we took leaf tissue from the injection area, extracted a fixed amount of total protein, and measured its fluorescence intensity.

Result shows that compared with NLR*+GFP group, GFP only and NLR+GFP had significantly higher fluorescence intensities, which indicates that our modified NLR* did produce effective clearance of GFP.

However, NLR+GFP also has a tiny reduction in fluorescence intensity compared to GFP only. We believe that this does not prove that unmodified NLR also has the ability to clear GFP, but can be explained by the increase in spontaneous dimerization after NLR overexpression.

Figure 2: Measurement of Fluorescence intensity in the leaves

Note:

NLR represents the original unmodified immune molecules.
NLR* represents an artificially modified molecule whose ID sequence has been replaced with a nanoantibody Enhancer targeting GFP

ROS Measurement

Reactive oxygen species (ROS) signaling plays an important role in the innate immune response of plants, can directly inhibit the growth of pathogens, and can also participate in the disease resistance process as a signaling molecule, and its rapid production is an important marker of plant defense system activation. ROS production is mainly mediated by NADPH oxidase, also known as Respiratory Burst Oxidase homologous Proteins (RBOHs) in plants, so plant ROS can be detected by elisa to determine the status and strength of the plant's immune response. We used the ROS kit (MM-43700M1) to experiment on plant tissues after 1 week of injection

Figure 3: Measurement of ROS content in the leaves

Note<p> <p>*P<0.05, ***P<0.001,****P<0.0001

NLR represents the original unmodified immune molecules.

NLR* represents an artificially modified molecule whose ID sequence has been replaced with a nanoantibody Enhancer targeting GFP.

As shown in the table and figure above, it can be seen that compared with normal tissue, 3 groups respectively injected with empty plasmids (1.807±0.067 vs. 1.1.716±0.004, P<0.05), GFP (1.803±0.045vs.1.716±0.004,P<0.05),and unmodified NLR+GFP(1.835±0.057 vs. 1.716±0.004, P<0.001) all had significant ROS elevations and remained similar levels of immunity. This can be explained by the plant's own immune response against Agrobacterium

The experimental group injected with the modified immune receptor + GFP showed a higher immune response intensity than the uninjected leaves (2.014±0.043 vs. 1.716±0.004, P< 0.0001). Even compared with other experimental groups (empty plasmid, GFP, unmodified NLR), there was a significant increase in ROS, indicating that our modified NLR by Agrobacterium transiently exactly enhanced the immune response of plant

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