Inhibitor activity of citrus polygalacturonase-inhibiting protein against the endopolygalacturonase of the oomyceteous pathogen Phytophthora parasitica

Our previous study has discovered a multigene family (pppg1-pppg10) encoding endopolygalacturonase (endoPG) in Phytophthora parasitica, an oomyceteous plant pathogen known to cause severe disease in a wide variety of plant species. They are induced in the process of plant infection, suggestive of their importance in the pathogenesis. Further analysis indicated that each PpPG plays a distinct role in the decomposition of plant cell wall. To find out if polygalacturonase-inhibiting proteins (PGIP) contain an inhibitor activity toward endoPGs from P. parasitica, we cloned PGIP genes from two Citrus species, Citrus grandis f. buntan Hay. and Citrus sinensis Osb.. Nucleotide sequences of PGIPs obtained from these two plants were identical, and we named one of the clones from C. grandis as cgpgip. Southern hybridization using cgpgip as a probe revealed the presence of a single copy gene in C. grandis, while two bands showed up in C. sinensis. Analysis of the deduced amino acid sequence indicated that CgPGIP contains a typical PGIP protein structure. Phylogenetic analysis indicated that PGIPs from Citrus spp. were clustered into a group distinct from PGIPs from Phaselous spp., which were well characterized and known to have an inhibitor activity toward endoPGs of fungi. To characterize the function of CgPGIP, recombinant proteins of CgPGIP and some PpPGs were expressed by using a yeast expression system, and used to analyze the inhibitor activity of CgPGIP. The results indicated that the recombinant protein of CgPGIP displayed an inhibitor activity toward PpPG2. Systemic expression of CgPGIP in Nicotiana benthamiana, by using the PVX agroinfection system, followed by inoculation with P. parasitica revealed that the CgPGIP-expressing plants are more resistant to this pathogen. Besides, co-expression of CgPGIP and PpPGs in planta resulted in a substantial reduction of the symptoms caused by PpPGs of group III, especially PpPG8 and PpPG10. These data suggest that, of the PpPGs of P. parasitica identified thus far, CgPGIP showed the inhibitor activity only toward PpPG2, PpPG8, and PpPG10, all of which belong to group III. Furthermore, expression of CgPGIP conferred certain degree of plant resistance against P. parasitica.