Table_1_Activation of Immune Genes in Leafhoppers by Phytoplasmas and Symbiotic Bacteria.docx

Insect immunity is a crucial process in interactions between host and microorganisms and the presence of pathogenic, commensal, or beneficial bacteria may result in different immune responses. In Hemiptera vectors of phytoplasmas, infected insects are amenable to carrying high loads of phytopathogens, besides hosting other bacterial affiliates, which have evolved different strategies to be retained; adaptation to host response and immunomodulation are key aspects of insect-symbiont interactions. Most of the analyses published to date has investigated insect immune response to pathogens, whereas few studies have focused on the role of host immunity in microbiota homeostasis and vectorial capacity. Here the expression of immune genes in the leafhopper vector of phytoplasmas Euscelidius variegatus was investigated following exposure to Asaia symbiotic bacteria, previously demonstrated to affect phytoplasma acquisition by leafhoppers. The expression of four genes related to major components of immunity was measured, i.e., defensin, phenoloxidase, kazal type 1 serine protease inhibitor and Raf, a component of the Ras/Raf pathway. The response was separately tested in whole insects, midguts and cultured hemocytes. Healthy individuals were assessed along with specimens undergoing early- and late-stage phytoplasma infection. In addition, the adhesion grade of Asaia strains was examined to assess whether symbionts could establish a physical barrier against phytoplasma colonization. Our results revealed a specific activation of Raf in midguts after double infection by Asaia and flavescence dorée phytoplasma. Increased expression was observed already in early stages of phytoplasma colonization. Gut-specific localization and timing of Raf activation are consistent with the role played by Asaia in limiting phytoplasma acquisition by E. variegatus, supporting the involvement of this gene in the anti-pathogen activity. However, limited attachment capability was found for Asaia under in vitro experimental conditions, suggesting a minor contribution of physical phytoplasma exclusion from the vector gut wall. By providing evidence of immune modulation played by Asaia, these results contribute to elucidating the molecular mechanisms regulating interference with phytoplasma infection in E. variegatus. The involvement of Raf suggests that in the presence of reduced immunity (reported in Hemipterans), immune genes can be differently regulated and recruited to play additional functions, generally played by genes lost by hemipterans.

昆虫免疫是宿主与微生物互作过程中的关键环节，病原性、共栖性或有益细菌的存在，均可引发不同的免疫应答。在植原体媒介半翅目(Hemiptera)昆虫中，受感染宿主不仅可携带高载量的植物病原菌，还会定殖其他附属细菌——这些细菌已演化出不同的滞留策略；对宿主免疫应答的适应与免疫调节，是昆虫-共生菌互作的核心环节。截至目前，已发表的多数研究聚焦于昆虫对病原菌的免疫应答，而针对宿主免疫在微生物群稳态及媒介效能中的作用的探讨较少。本研究以植原体媒介昆虫斑叶蝉(Euscelidius variegatus)为对象，探究其暴露于共生菌阿萨亚菌(Asaia)后的免疫基因表达情况；此前已有研究证实，该菌可影响叶蝉对植原体的获取能力。本研究检测了4种与免疫核心组分相关的基因的表达水平，分别为防御素(defensin)、酚氧化酶(phenoloxidase)、Kazal型1丝氨酸蛋白酶抑制剂(Kazal type 1 serine protease inhibitor)以及Ras/Raf通路组分Raf蛋白(Raf)。实验分别在完整虫体、中肠以及体外培养的血淋巴细胞中检测了免疫应答情况，同时评估了健康个体，以及处于植原体感染早期和晚期阶段的标本的免疫基因表达。此外，本研究还检测了阿萨亚菌菌株的黏附等级，以探究共生菌是否可通过形成物理屏障阻碍植原体的定殖。本研究结果显示，在同时感染阿萨亚菌与金黄褪绿植原体(flavescence dorée phytoplasma)后，斑叶蝉中肠内的Raf蛋白发生了特异性激活，且早在植原体定殖的早期阶段，即可检测到Raf表达水平的升高。Raf激活的肠道特异性定位与时间进程，与阿萨亚菌对斑叶蝉获取植原体的限制作用相符，证实了该基因参与了抗病原菌免疫活动。但在体外实验条件下，阿萨亚菌的黏附能力有限，这表明共生菌通过物理屏障阻碍植原体黏附媒介肠道壁的作用较为微弱。本研究证实了阿萨亚菌的免疫调节作用，相关结果有助于阐明斑叶蝉体内调控植原体感染干扰过程的分子机制。Raf蛋白的参与表明，在半翅目昆虫已被报道的免疫功能弱化状态下，免疫基因可被差异化调控并募集以执行额外功能——这类功能通常由半翅目昆虫已丢失的基因所承担。