青枯病菌效應蛋白 PopP3 抑制植物防禦之機制研究

Abstract

青枯病菌 (Ralstonia solanacearum, Rs) 所引發之青枯病為極重要土壤傳播性植物細菌病害，常造成全球許多高經濟農作物重大損失。Rs 菌系極為複雜，且屬於phylotype I 之 Rs 菌株在番茄之毒力亦有差異性，然而目前關於 Rs 毒力之分子機制瞭解仍非常有限。本研究室先前發現高毒力 Rs 菌株 Pss190 特有的效應蛋白 Pseudomonas outer protein P 3 (PopP3)，在植物中過量表現 popP3 時會壓抑植物之pathogen/microbe-associated molecular patterns (PAMP/MAMP)-triggered immunity (PTI)。本研究旨在進一步探討 PopP3 抑制植物防禦反應之分子調控機制。定序結果發現同為穩定高毒力菌株之 Pss190 與 Pss365 其 PopP3 序列完全相同，支持 PopP3 與 Rs 高毒力之可能相關性。培養基試驗顯示 PopP3 確實可被分泌至 Rs 細胞外，符合微生物效應蛋白特性。PopP3-GFP 會以點狀分佈在圓葉菸草葉細胞質與細胞核中。在穩定抗 Rs 之番茄品系 Hwaii 7996 (H7996) 中表現 PopP3 會減低在 PTI 具關鍵正向調控者功能 之 12g520 的轉錄體量與其調控之 ROS burst，且初步結果指出 12g520 可能與 PopP3 具交互作用。此外，表現 PopP3 會減低誘導 PTI 後之乙烯合成相關標誌基因 ACO1 的轉錄體量。在圓葉菸草表現 PopP3 並透過共免疫沉澱與液相層析串聯二次質譜(Co-IP-LC-MS/MS) 技術篩選 PopP3 之可能互作蛋白質，其中已知參與植物防禦反應之 CALCIUM-SENSING RECEPTOR (CAS) 與已知參與青枯病抗性之SESQUITERPENE SYNTHASE (TPS1) 會提升 PTI 相關之 ROS burst，而同時表現 PopP3 會減低 CAS 或 TPS1 所提升之 ROS 總量，且初步結果指出 CAS 與 TPS1可能與 PopP3 具交互作用。預期這些研究結果將有助於闡明 PopP3 影響植物病害免疫力之具體調控機制。

Ralstonia solanacearum (Rs) is a soil-borne vascular plant pathogen, causing deadly bacteria wilt (BW) and thus serious economic losses on a wide range of crops worldwide. Rs strains are highly complex and phylotype I strains confer varied degrees of virulence/aggressiveness on tomato plants; however, genetic information on pathogen virulence-related factors is still very limited. Our previous studies revealed that a predicted YopJ-type effector protein, Pseudomonas outer protein P 3 (PopP3), is unique to the high-virulence strain Pss190, and PopP3 reduces pathogen/microbe-associated molecular patterns (PAMP/MAMP)-triggered immunity (PTI) in tomato and Nicotiana benthamiana. This study aimed to further elucidate the involved mechanisms. Results of sequencing assay revealed that PopP3 sequences in Pss190 and Pss365 are identical, supporting that PopP3 could be crucial for high-virulence of Rs. Medium secretion assay confirmed that PopP3 is secreted by Rs. When expressed in N. benthamiana leaf, PopP3-GFP forms cytosolic and nuclear foci. Expression of PopP3 in tomato reduces the transcript level of 12g520, a gene with a crucial positive role in tomato PTI, and 12g520-mediated PTI-related ROS burst. Preliminary data further suggested possible interaction between PopP3 and 12g520 protein. Expression of PopP3 also reduced the transcript level of ethylene biosynthesis enzyme ACO1, after triggering PTI. Furthermore, Co-IP-LC-MS/MS was used to search for interacting plant proteins of PopP3 in N. benthamiana. Among the candidate interacting proteins, CALCIUM-SENSING RECEPTOR (CAS) and SESQUITERPENE SYNTHASE 1 (TPS1) play a positive role in PTI-related ROS burst, and expression of PopP3 reduces this effect. Preliminary data further suggested possible interactions between PopP3 and these two proteins. These results are expected to help elucidate the exact mechanism underlying the PopP3’s suppressing effect on plant PTI.