探討柑橘多聚半乳糖醛酸酶抑制蛋白對疫病菌多聚半乳糖醛酸酶的抑制作用

Abstract

植物抵抗病原菌侵染所涉及的機制相當複雜，而植物細胞間隙為兩者攻防的重要場域之一。許多植物病原菌會外泌細胞壁分解酵素破壞植物細胞壁，其中的多聚半乳糖醛酸酶(endopolygalacturonase, endoPG)被認為是感染初期最早產生的酵素之一。植物的多聚半乳糖醛酸酶抑制蛋白(polygalacturonase-inhibiting protein, PGIP)對真菌的endoPG有抑制作用，並參與在長鏈寡半乳糖醛酸(long-chain oligogalacturonides, OGs)誘導植物抗病的訊息傳導途徑中，於植物抵抗病原菌的致病過程扮演重要角色。Phytophthora parasitica為重要的植物病原卵菌，本實驗室先前的研究自其基因體選殖出十個endoPG基因(pppg1-pppg10)，這些基因在P. parasitica侵染植物過程會被誘導表現，且個別於菸草(Nicotiana benthamiana)進行系統性表現時所造成的影響各有不同，顯示其在分解植物細胞壁時各扮演獨特的角色。為了解植物PGIP在植物抵抗卵菌與其對卵菌endoPG的抑制作用，我們從P. parasitica的寄主植物Citrus grandis f. buntan Hay.與Citrus sinensis Osb.中選殖PGIP基因。由序列分析結果得知，與其他植物PGIP具同源性，命名為cgpgip。南方雜合分析顯示C. grandis僅含單一個PGIP基因，但在C. sinensis基因體中應有其他cgpgip的同源性序列存在。親緣分析的結果顯示，PGIP依植物所屬分科而分群，且柑橘與其他芸香科的PGIP有較高的同源性。利用甲醇酵母菌(Pichia pastoris)表現CgPGIP與PpPG的重組蛋白，並對CgPGIP抑制PpPG活性情形進行分析，發現對CgPGIP對PpPG2有抑制效果。以PVX agroinfection在菸草上系統性表現CgPGIP，隨後再挑戰接種P. parasitica，結果發現表現CgPGIP之植物的發病程度比較輕微。此外，以PVX agroinfection在菸草同時表現PpPG與CgPGIP，觀察PpPG所造成的症狀受到CgPGIP影響情形時發現，CgPGIP會減弱被歸類為第三群的PpPG所造成的效應，此現象在針對PpPG8和PpPG10特別顯著，但在第二群與第四群PpPG則見不到此情形，顯示CgPGIP對於個別PpPG的抑制作用有選擇性。

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.