探討疫病菌β-1,3-glucanase基因在植物-疫病菌交互作用的角色

Investigate the role of a β-1,3-glucanase encoding gene in Phytophthora parasitica-plant interaction

江秉真(Bing-Jen Chiang)

指導教授 ： 劉瑞芬

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202202821

摘要

在植物與病原菌交互作用中，植物病原菌會分泌許多細胞壁分解酵素 (cell wall degrading enzymes, CWDEs)，以分解或弱化植物細胞壁的結構幫助入侵。近年研究發現在與病原菌長年共演化過程中，植物可藉由辨識部分CWDEs為pathogen-associated molecular patterns (PAMPs)或其分解產物[稱為damage-associated molecular patterns (DAMPs)]引發植物pattern-triggered immunity (PTI)。疫病菌質外體效應蛋白OPEL及其同源性基因可在菸草引發PTI反應，包括細胞死亡、癒傷葡聚醣沉積、活性氧分子累積及誘導防禦基因表現等。OPEL及其同源性基因的GH16 domain被預測具有β-1,3-聚葡萄糖酶保守性序列，而且其突變嚴重影響OPEL誘發植物防禦反應的能力，顯示β-1,3-聚葡萄糖酶活性可能在疫病菌與植物的交互作用扮演重要角色。為探討這個可能性，本研究自疫病菌挑選5個編碼β-1,3-聚葡萄糖酶、屬於GH17基因族的候選基因，並以agroinfection在圓葉菸草過表現，發現其中的PPG24在系統葉造成明顯黃化、嵌紋、葉片皺縮等病徵。PPG24僅存在於卵菌，在細菌、真菌、動物與植物皆未發現其同源基因。在疫病菌感染植物後3小時，PPG24即大量表現，顯示其極有可能作用於疫病菌感染植物前期。此外，在過表現PPG24的圓葉菸草離葉接種疫病菌可促進病害發展。為探討PPG24在疫病菌致病過程的重要性，本研究建立以”rub-induced gene silencing”靜默PPG24的技術，發現疫病菌在PPG24 5-UTR dsRNA處理植物之系統葉的致病力顯著降低；以共軛焦顯微鏡觀察，發現在這些葉片之疫病菌靜止子所長出的發芽管有部分明顯變短，顯示PPG24可能在靜止子萌發後的發芽管延伸過程扮演關鍵角色，從而影響疫病菌的致病力。另一方面，過表現PPG24可誘導PAL以及Pti5基因的表現，但顯著降低Potato virus X (PVX)的累積量；被刪除訊息胜肽的PPG24突變蛋白完全失去引發前述病徵的活性，卻仍保有減少PVX累積的能力。這些結果顯示PPG24必須被分泌至質外體，才能在植物引發嚴重病徵，可是這病徵並非導因於病毒的大量增殖。此外，不論是否含帶訊息胜肽，PPG24都能調降PVX的累積量，相關調控機制仍待進一步探討。

關鍵字

疫病菌 ； β-1,3-聚葡萄醣酶 ； GH17 ； 基因靜默 ； 細胞壁分解酵素

並列摘要

Upon infection of plants, pathogens secret various cell wall degrading enzymes (CWDEs) to help degrade or weaken cell wall to facilitate infection. During the co-evolution with pathogens, plants have evolved the ability to recognize some of the CWDEs as pattern-associated molecular patterns (PAMPs) or their degrading products as damage-associated molecular patterns (DAMPs) to trigger pattern-triggered immunity (PTI). Phytophthora parasitica apoplastic effector OPEL can trigger PTI responses including cell death, callose deposition, ROS production, and expression of defense genes in Nicotiana tabacum (cv. Samsun-NN). The glycoside hydrolase 16 (GH16) domain of OPEL contains conserved signature of β-1,3-glucanase, which is indispensable for OPEL to trigger PTI response. To know whether other β-1,3-glucanase-encoding genes of P. parasitica such as those of the GH17 family show similar activity, five GH17 candidate genes were selected and assayed by agroinfection of Nicotiana benthamiana. One of these genes, named PPG24, triggered severe symptoms of yellowing, mosaic, and crinkling on systemic leaves of N. benthamiana. Homologs of PPG24 exist only in oomycetes, but not in bacteria, fungi, plants, and animals. The expression of PPG24 was highly induced at 3 and 6 hour post infection of P. parasitica. When overexpressed, PPG24 enhanced disease severity caused by P. parasitica. To verify the importance of PPG24 in virulence, a “rub-induced gene silencing” method was established. On the systemic leaves of N. benthamiana plants pretreated with PPG24 5-UTR dsRNA, virulence of P. parasitica was significantly compromised. Moreover, the length of germ tubes of some germinating cysts became much shorter than that on the control plants. Therefore, PPG24 appears to be a key factor required for the elongation of germ tubes and thereby is involved in the early infection stage of P. parasitica. On the other hand, PPG24 overexpression upregulated the expression of PAL and Pti5, whereas downregulated PVX accumulation. Notably, PPG24 devoid of signal peptide failed to cause the aforementioned severe symptoms, but retained part of the PPG24 activity to downregulate PVX accumulation. These results indicate secretion to the apoplast is essential for PPG24 to induce severe symptoms, which is less likely caused by increased accumulation of viruses though. As well, no matter containing signal peptide or not, PPG24 overexpression downregulated PVX accumulation. Further analysis will help to elucidate the underlying mechanism.

並列關鍵字

Phytophthora parasitica ； β-1,3-glucanase ； GH17 ； gene silencing ； cell wall degrading enzyme

參考文獻

Ahn, S. J., Donahue, K., Koh, Y., Martin, R. R., and Choi, M. Y. 2019. Microbial-based double-stranded RNA production to develop cost-effective RNA interference application for insect pest management. Int. J. Insect Sci. 11:1179543319840323.

Amaro, T. M., Thilliez, G. J., Motion, G. B., and Huitema, E. 2017. A perspective on CRN proteins in the genomics age: evolution, classification, delivery and function revisited. Front. Plant Sci 8:99.

Aslam, S. N., Erbs, G., Morrissey, K. L., Newman, M. A., Chinchilla, D., Boller, T., Molinaro, A., Jackson, R. W., and Cooper, R. M. 2009. Microbe-associated molecular pattern (MAMP) signatures, synergy, size and charge: influences on perception or mobility and host defence responses. Mol. Plant Pathol. 10:375-387.

Böhm, H., Albert, I., Oome, S., Raaymakers, T. M., Van den Ackerveken, G., and Nürnberger, T. 2014. A conserved peptide pattern from a widespread microbial virulence factor triggers pattern-induced immunity in Arabidopsis. PLoS Pathog. 10:e1004491. doi: 10.1371/journal.ppat.1004491.

Baek, E., Yoon, J. Y., and Palukaitis, P. 2017. Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco. Virology 510:29-39.