植物病原菌Pseudomonas syringae pv. tomato DC3000中PSPTO_0281 基因之特性分析

Abstract

Pseudomonas syringae為一具極性鞭毛之革蘭氏桿狀菌，由於其寄主範圍廣泛且其寄主多具經濟價值，它已成為被研究最多的模式植物病原細菌之一。過去的研究指出，其病原性及致病力主要是由第三型分泌系統及其分泌之效應蛋白所掌控；然而我們的研究初步發現，第六型分泌系統在Pto DC3000 與其他微生物的競爭能力上扮演重要的角色，進而影響其在生態上的適應性。為了進一步了解第六型分泌系統所調控的功能，我們針對Pto DC3000第第六型分泌系統基因叢集 (gene cluster) 內的轉錄因子，PSPTO_5424 (sfa2)，進行了次世代轉錄體定序的分析，結果發現Sfa2僅調控少數基因，其中一個受到 Sfa2負向調控的基因為PSPTO_0281。PSPTO_0281的基因產物可能是 H-NS (Histone like nucleoid structuring protein)，推測可能為一轉錄抑制蛋白，可廣泛調控多種基因的表現，尤其是外來基因的表現情形。從qRT-PCR、GUS 活性測試與西方免疫墨點法的結果發現H-NS對hcp2基因或其蛋白質的表現與分泌並沒有太大的影響。但是在競爭實驗中，當大量表現PSPTO_0281時，Pto DC3000 對 E. coli MG1655及 P. syringae pv. phaseolicola 1448a的競爭能力會稍微降低。此外，前人研究發現第三型分泌系統與第六型分泌系統之間的調控機制往往伴隨著相互制衡的現象。因此我們進而探討PSPTO_0281與Sfa2是否對Pto DC3000第三型分泌系統有影響。在即時反轉錄聚合酶連鎖反應與GUS 活性測試的結果發現第三型分泌系統重要調控因子hrpR 與hrpL及效應蛋白之基因 avrPto的表現量在Δsfa2突變株及Δsfa2Δ0281雙突變株中均有升高的趨勢，顯示PSPTO_0281 和Sfa可能扮演負調控第三型分泌系統的角色。另一方面，Pto DC3000的病原性分析實驗中顯示，Δsfa2突變株或大量表現 PSPTO_0281 的菌株對於番茄的致病能力比野生株高，但在菸草中幾乎完全相反。這也表示了在不同的宿主植物中，病原菌所引起的反應可能有所差異。最後，我們也證明了 PSPTO_0281 確實是一個DNA 結合蛋白，然而與其結合的 DNA 序列並無專一性可。

Pseudomonas syringae is a rod-shaped, Gram-negative bacterium with polar flagella. As a plant pathogen with broad host range and economical importance, P. syringae has become one of the most studied model phytopathogen. From previous studies, we have known that type III secretion system (T3SS) plays a crucial role in the pathogenicity of P. syringae. In addition, the type VI secretion system (T6SS) has been shown to be involved in the fitness of interbacterial competition. By means of RNA sequencing to identify gene differentially expressed in P. syringae pv. tomato DC3000 wild type and Δsfa2, we discovered one gene, PSPTO_0281, which was highly expressed in Δsfa2. Although PSPTO_0281 was predicted to encode a putative histone-like nucleoid structuring (H-NS) protein, whether it acts as a transcriptional regulator remain to be elucidated. Using qRT-PCR, GUS activity and Western blot analysis, the expression and secretion of hcp2 were found independent of PSPTO_0281. However, overexpression of PSPTO_0281 slightly decreased the interbacterial competition ability of Pto DC3000 against E. coli MG1655 and P. syringae pv. phaseolicola 1448a. Considering the reciprocal regulation of T6SS and the T3SS in the gene regulatory network of Pto DC3000, T3SS-related genes were further analyzed. Indeed, PSPTO_0281 as well as Sfa2 both negatively regulate the expression of T3SS regulators and effectors, including hrpR, hrpL and avrPto. In the pathogenicity assay, overexpression of PSPTO_0281 enhanced virulence in Pto DC3000 as shown in Δsfa2 mutant. However, different results were obtained when Nicotiana benthamiana was used as host plants, suggesting different mechanisms were deployed in different plants in response to pathogen attack. Lastly, we also verified that PSPTO_0281 encodes a DNA-binding protein, which bind to DNA nonspecifically.