Cloning, expression analysis and recombinant expression of a gene encoding a polygalacturonase-inhibiting protein from tobacco, Nicotiana tabacum

Abstract

Polygalacturonase inhibiting proteins (PGIPs) are major defensive proteins produced by plant cell walls that play a crucial role in pathogen resistance by reducing polygalacturonase (PG) activity. In the present study, a novel PGIP gene was isolated from tobacco (Nicotiana tabacum), hereafter referred as NtPGIP. A full-length NtPGIP cDNA of 1,412 bp with a 186 bp 5′-untranslated region (UTR), and 209 bp 3′-UTR was cloned from tobacco, NtPGIP is predicted to encode a protein of 338 amino acids. The NtPGIP sequence from genomic DNA showed no introns and sequence alignments of NtPGIP’s deduced amino acid sequence showed high homology with known PGIPs from other plant species. Moreover, the putative NtPGIP protein was closely clustered with several Solanaceae PGIPs. Further, the expression profile of NtPGIP was examined in tobacco leaves following stimulation with the oomycete Phytophthora nicotianae and other stressors, including salicylic acid (SA), abscisic acid (ABA), salt, and cold treatment. The results showed that all of the treatments up-regulated the expression of NtPGIP at different times. To understand the biochemical activity of NtPGIP gene, a full-length NtPGIP cDNA sequence was subcloned into a pET28a vector and transformed into E. coli BL21 (DE3). Recombinant proteins were successfully induced by 1.0 nmol/L IPTG and the purified proteins effectively inhibited Phytophthora capsici PG activity. The results of this study suggest that NtPGIP may be a new candidate gene with properties that could be exploited in plant breeding.

Direct evidence for a new mode of plant defense against insects via a novel polygalacturonase-inhibiting protein expression strategy

“…However, studies on heterologous expression of PGIPs, apart from in planta, are scarce and limited to few candidate proteins. Some PGIPs were successfully expressed in Escherichia coli (39,42,44,49), whereas other studies report on their accumulation into inclusion bodies (14,50). When studying novel PGIPs, assessment of whether renaturation restores the native function is difficult, because the target PG is unknown.…”

Recent Advances in Understanding the Function of the PGIP Gene and the Research of Its Proteins for the Disease Resistance of Plants

“…The PGIP anti-disease effect has been confirmed in a series of in vivo experiments. PGIP-mediated fungi resistance has been shown in various important cash crops, such as grapes [57,58], papaya [59], potatoes [51], alfalfa [15], cotton [60], green beans [6,28,61], tomatoes [62], peas [63], mandarin orange [64], tobacco [65], and Arabidopsis [66]. PGIP expression does not affect plant phenotype or inhibit plant growth in sugar beet [67] or tobacco [67].…”

Elucidating bacterial adhesion to mucosal surface by an original AFM approach

“…The sialic acids can be utilized by pathogens as nutrients [4] or attachment sites for bacterial adhesion [5]. The study of sialic acid binding adhesins has revealed similar lysine-rich sialic acid binding motifs in lectins on Escherichia coli and Helicobacter pylori [6,7]. A large diversity in mucin glycosylation is observed between species, and also between organs within the same animal [8].…”