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

Abstract

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.

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

“…This provides indirect evidence that PGs are important pathogenic factors in the infection of Arabidopsis by the two fungal pathogens. Previous studies have reported the effectiveness of PGIP in reducing the disease symptoms in other transgenic plants infected with B. cinerea and Cercospora nicotianae 1841. Here, we provide evidence that GhPGIP1 gene can also protect Arabidopsis against infection by the two fungi.…”

“…Firstly, top 20 solutions of the complexes were generated by PatchDock, according to the scoring function used. Subsequently, FireDock was used for the fast interaction refinement of the top 10 complexes that were rescored according to the criteria described previously41. Then the prediction candidate models were re-ranked due to re-refinement and optimization (Supplementary Table 1).…”

“…A PolyATract mRNA Isolation System was used to obtain polyadenylated mRNA following the specifications of the supplier (Promega). Thereafter, a cDNA library was constructed as described previously4162, based on a 21-amino acid sequence (5′-FDXSYFHNKCLCGAPLPSCK-3′) conserved at the C-terminus of all the previously characterized PGIPs915.…”

“…The culture was further incubated for 6 h at 28 °C and was harvested, thereafter, by centrifugation at 10,000 ×  g for 20 min at 28 °C. The expression of soluble GhPGIP1 was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the recombinant protein was purified using a 6× His-Tagged Protein Purification Kit (CW BIO)41. The 6× His fusion tags, trx∙tag and S∙tag, were removed using a Thrombin Cleavage Capture Kit (Novagen, Madison, WI).…”

“…The plants were then challenged with a conidial suspension of V. dahliae and F. oxysporum f. sp. vasinfectum by syringe inoculation as reported earlier41. The disease lesion areas were measured after 3–5 d. All the disease assays were repeated at least four times.…”