探討NtWAKL14在OPEL引發之植物防禦反應的角色

Abstract

Wall-associated receptor kinases (WAKs) 是植物特有的一群receptor-like protein kinases (RLKs)，其結構包含位於胞外的WAK-GUB域與EGF域、跨膜域以及胞內的蛋白激酶域。WAKs與序列相似的WAK-likes (WAKLs) 為龐大的基因家族，部分成員作為植物細胞壁果膠受體，和細胞擴展、植物發育有關；部分則與植物防禦反應有關，在植物受病原入侵等特定刺激時被誘導表現，但不同植物的WAKs與WAKLs參與防禦反應的機制不盡相同。雖然WAKs與WAKLs已被證實參與多種植物對真菌及細菌病原的抗性，其在植物與疫病菌交互作用的角色亟待探究。Phytophthora parasitica質外體效應蛋白OPEL可在菸草Nicotiana tabacum (cv. Samsun-NN) 引發癒傷葡聚醣沉積、活性氧分子累積以及防禦相關基因表現。分析菸草以OPEL重組蛋白處理後的轉錄體，發現多個WAK與WAKL基因表現量顯著提升。為探討這些基因在OPEL引發植物免疫反應以及植物－疫病菌交互作用的角色，本研究首先進行序列分析，發現這些菸草WAKs與WAKLs大多具有典型的WAK蛋白結構；親緣分析結果顯示菸草除具有阿拉伯芥WAKs的直系同源性基因 (orthologs) 外，還含有其他數個支序群。qRT-PCR分析發現處理OPEL重組蛋白與接種P. parasitica可誘導特定菸草WAK與WAKL候選基因的表現，包括NtWAKL14x1與NtWAKL14x2。於圓葉菸草暫表現NtWAKL14x1-GFP，發現其位於植物細胞膜。藉由cucumber mosaic virus (CMV) 於Nicotiana tabacum (cv. Samsun-NN) 誘導靜默NtWAKL14x1與NtWAKL14x2，可降低OPEL引發之癒傷葡聚醣沉積與細胞死亡等植物防禦反應，並促進疫病菌感染。這些結果顯示NtWAKL14x1與NtWAKL14x2正向調控OPEL引發的防禦反應，也是首次發現WAKL參與植物對卵菌的抗性，其詳細作用機制有待後續探究。

Wall-associated receptor kinases (WAKs), a subfamily of receptor-like protein kinases (RLKs) found only in plants, consist of extracellular WAK-GUB and EGF domain, transmembrane domain, and intracellular kinase domain. WAKs and its homologs, known as WAK-likes (WAKLs), play diverse roles in plants. Some WAKs associate with pectin in plant cell wall and take part in the regulation of plant cell expansion and development, whereas some are involved in defense responses through various mechanisms. Although the roles of WAKs and WAKLs in plant interactions with fungal and bacterial pathogens have been well studied, their contribution to plant resistance against the oomycetes remains largely unknown. OPEL, an apoplastic effector from Phytophthora parasitica, can induce callose deposition, ROS production, and defense gene expression in Nicotiana tabacum (cv. Samsun-NN). Previous study based on transcriptome analysis found several NtWAKs and NtWAKLs were significantly induced by OPEL treatment. Here, we further characterized these genes to investigate their roles in plant immune responses toward OPEL and P. parasitica. Sequence analysis indicates most of these candidate genes contain predicted protein structure similar to that of typical WAKs. Phylogenetic analysis showed some of these NtWAKs are orthologous to AtWAKs from Arabidopsis thaliana, whereas others form distinct clades. Analysis by qRT-PCR demonstrated the induction of specific NtWAKs and NtWAKLs after OPEL treatment and P. parasitica inoculation, including NtWAKL14x1 and NtWAKL14x2. When overexpressed on Nicotiana benthamiana, NtWAKL14x1-GFP localized in the plasma membrane. Downregulation of NtWAKL14x1 and NtWAKL14x2 by cucumber mosaic virus (CMV)-mediated gene silencing compromised OPEL-induced callose deposition and cell death, whereas enhanced P. parasitica infection on Nicotiana tabacum (cv. Samsun-NN). These results indicate NtWAKL14x1 and NtWAKL14x2 function as positive regulators of plant defense response toward OPEL and P. parasitica. This is the first report of WAKL genes to participate in the plant-oomycete interaction. Further investigations are needed for better understanding of the underlying mechanism.