第三型作用蛋白AvrPtoBB728a 誘導番茄抗性功能區域之分析

Abstract

Pseudomonas syringae 為革蘭氏陰性菌，已知可感染許多植物造成病徵，由於其廣大的寄主範圍及經濟上的重要性，使其成為被廣泛研究最多的植物病原菌之一。P. syringae 在進入植物後會藉由第三型分泌系統，將其作用蛋白直接送到植物細胞內，藉由干擾植物正常生理機能或抑制植物先天免疫系統，使病原菌成功在細胞中生長繁殖，最後造成植物產生病徵。植物在演化的過程中，產生能夠辨認作用蛋白的抗性基因(resistance gene)，進一步啟動更專一的寄主免疫反應，成功地阻止病原菌生長。AvrPtoB 為P. syringae pv. tomato 中會經由第三型分泌系統送到植物細胞中的作用蛋白之一，可被抗性基因產物辨認，啟動下游訊息傳導而引發抗性反應。之前研究發現豆類病原菌P. s. pv. syringae B728a 的AvrPtoB 的同源蛋白 (稱為AvrPtoBB728a)，除了能誘發 Pto 抗性外，在沒有 Pto 的番茄中，仍能誘發抗性保護植物；而此抗性已證明與番茄Pto 基因座中的Pto 基因家族及prf 有關。因此，我的研究主要目的為：找出在蕃茄中負責辨認AvrPtoBB728a、誘發專一性抗性的植物基因；此外，也配合功能分析法，將分成大小不同區塊的AvrPtoBB728a，利用酵母菌雙雜交法及病原性測試法，確認對AvrPtoBB728a 功能重要的區塊。利用 此策略，我們成功地找出SlPtoB 為負責此番茄抗性的基因，也進一步得到能與誘發此抗性所需的AvrPtoBB728a 作用區塊，此結果證明了在抗性基因座上的同源基因確實有其抗性功能，而非僅是單純基因複製的結果。此外，AvrPtoBB728a 的蛋白質序列雖然與 AvrPtoB 相似，其作用卻不盡相同，可能是透過不同的機制來引發不同番茄品種的抗性。未來可針對已知的AvrPtoB 特性，來探討AvrPtoBB728a 之所以功能不同的原因。

Pseudomonas syringae, a Gram-negative rod-shaped bacterium, can infect a wide array of host plants and cause different disease symptoms. Due to its broad host range and economical importance, P. syringae has become one of the most studied model organisms. After P. syringae enters plants, various effector proteins can be delivered into host cells via type III secretion system, which can then interfere with normal physiological state or inhibit basal defense responses of plants, leading to colonization and growth of the pathogens, and later, disease symptoms on host plants. However, plants have evolved some resistance genes that can recognize these effectors to activate specific host resistance in order to block the bacterial growth. AvrPtoB, a well-known type III effector of P. syringae pv. tomato, is recognized by a resistance protein Pto to activate downstream signal transduction for resistance responses. Like AvrPtoB, a homolog of avrPtoB from a bean pathogen P. syringae pv. syringae B728a(AvrPtoBB728a), can trigger Pto-mediated resistance, but can also induce resistance on cultivate tomato lacking Pto. Previous studies have proved that this resistance is conferred by Pto family members and Prf. Thus, the purpose of this study was to identify plant genes responsible for this host resistance, and domains in AvrPtoBB728a required for this host resistance. Using a yeast two-hybrid system combined with the pathogenicity assay, we successfully identified that SlPtoB was responsible for tomato resistance and found out the smallest region of AvrPtoBB728a required for recognition is amino acids 91-330. These results proved that Pto paralogs in Pto locus do have resistance function, not simply a phenomenon of gene duplication. AvrPtoBB728a shares high sequence similarity with AvrPtoB, but they have quite different functions. This may imply that they activate tomato resistance mechanisms via different pathways. In the future, features of AvrPtoB can be focused for investigation of the reason why AvrPtoBB728a functions differently from AvrPtoB.