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PehA

PehA is a pathogenic factor of host plant infection secreted by R. solanacearum. Its essence is pectin endonuclide, that is, multi-galacturonic endonuclide, which can hydrolyze pectin, an important component of host plant cell wall, and produce Oligogalacturonides (OGs), thereby improving the infection ability of R. solanacearum. Host plant cells evolved WAK1, a cell membrane surface pattern recognition receptor that recognizes OGs, which is a Damaged-associated molecular pattern (DAMP), and thus stimulates a typical PTI immune response.

Sequence and Features

Biology

The bacterial wilt pathogen Ralstonia solanacearum produces three extracellular polygalacturonases (PGs): PehA, PehB, and PehC. All three PGs hydrolyze pectin's polygalacturonic acid backbone, but each releases different reaction products. PG is related to fruit maturation, cell separation processes (such as leaf and flower shedding, pod cracking, pollen maturation, pathogen defense, plant host interaction), and cell extension, development, and lignification. Therefore, PG has always been a focus of research on plant development and fruit maturation and senescence.

Design

We designed exogenous expression of PehA, a polygalacturonic endonuclidenase, and then used it to treat plant cell wall or pectin polysaccharide. After obtaining OGs of oligogalacturonic acid as a hydrolyzed product, plant tissues were treated to detect whether it had an immune reaction, which was used as an immune inducible factor for our project. We obtained the amino acid sequence of PehA of R. solanacearum in literature, conducted blastp, and obtained two results. We compared the sequence on snapgene, and then compared it with the CDS sequence of PehA found in NCBI database, and selected the one with high matching degree.

Plasmid construction

The obtained target gene sequence was assembled into the polyclonal site of the pGS-21a plasmid, and the GST protein purification tag sequence was linked to facilitate subsequent protein extraction and purification experiments. The recombinant plasmid was transferred into the proteinase-deficient Escherichia coli BL21 (DE3), and the fermentation of PehA was induced by IPTG.

Functional verification

In the experiment, we tried to induce the expression of PehA by Escherichia coli, and then separated and purified it and digested pectin. The product obtained was oligogalacturonic acid. Plant leaves were treated with OGs, and immunoresponse-related indicators were then tested to verify effectiveness. However, due to the short experimental time and limited equipment conditions, we gradually gave up the oligosaccharide production program, but we still chose to record our experimental attempts to provide experience for iGEM teams who choose plant immune-related content in the future.

Recombinant plasmid electrophoresis

In our experiment, the target protein was extracted and purified according to the protein label and treated to the leaves of tomato. BvEP can stimulate the outbreak of reactive oxygen species in tomato leaves as an immune response. Hydrogen peroxide, a kind of ROS, can be used to calculate or predict the degree of immune response by observing or counting the relative gray value of leaves after the brown red precipitates generated by DAB staining are decolorized by ethanol.

Expression of Proteins

The expression of immune protein factors is regulated by lactose operon, so IPTG is applied to induce our protein expression, and PehA protein is extracted and purified by GST tag protein purification kit. The protein concentration was determined by the BCA method. We controled the experiment and found that protein expression levels were higher when induced at 100 rpm in a shaker at 25 ° C. The expression concentration of PehA protein reached 0.199 mg/ml.

Due to the short experimental time and limited equipment conditions, we gradually gave up the oligosaccharide production program, but we still chose to record our experimental attempts to provide experience for iGEM teams who choose plant immune-related content in the future.

Reference

[1] Kohorn B D, Kohorn S L. The cell wall-associated kinases, WAKs, as pectin receptors[J]. Frontiers in plant science, 2012, 3: 88. [2] Ferrari S, Savatin DV, Sicilia F, Gramegna G, Cervone F, Lorenzo GD. Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development. Front Plant Sci. 2013 Mar 13;4:49. doi: 10.3389/fpls.2013.00049. [3] Huang Q, Allen C. Polygalacturonases are required for rapid colonization and full virulence of Ralstonia solanacearum on tomato plants[J]. Physiological and molecular plant pathology, 2000, 57(2): 77-83. [4] Huang Q, Allen C. Polygalacturonases are required for rapid colonization and full virulence of Ralstonia solanacearum on tomato plants[J]. Physiological and molecular plant pathology, 2000, 57(2): 77-83. [5] Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development