A host-derived chimeric peptide protects citrus against Huanglongbing without threatening the native microbial community of the phyllosphere

The application of host-derived antibacterial peptides has been highlighted as a potential efficacious and safe tool for treatment of Huanglongbing (HLB), the most devastating disease of citrus. However, pathogenic bacteria such HLB-causing Candidatus Liberibacter asiaticus (CLas) often develop resistance against the host antibacterial peptides. We showed that chimeras containing two different host antibacterial peptides not only retain antibacterial activity but also overcome bacterial resistance and enhance plant defense responses. Also, chimeric peptides can have an off-target impact on the structure and function of plant associated microbiome. However, there is a lack of understanding of the impact of the chimeric peptide therapy on the microbial structure in the citrus phyllosphere while reducing the CLas titers. Here, we aim to evaluate the efficacy of a chimeric peptide (UGK17) to reduce CLas titers, inducing plant defense response and impacting the microbiome associated with the citrus phyllosphere. Leaf samples were collected from orange and grapefruit trees in Texas and identified as old and young leaves according to their maturity. We collected three different types of leaves based on their infection and symptoms: healthy, symptomatic (infected with typical symptoms), and asymptomatic (infected s without symptoms). In planta assay was performed by dipping the leaves in the 0, 5, and 25 uM of UGK17 solutions for 48 hours. The quantifications of CLas titer and pathogenesis-related (PR) gene expression were done by qPCR and RT-qPCR, respectively. Amplicon sequencing was done to evaluate the impact of UGK17 on individual bacterial community structure. In addition, we performed an ex planta assay to access the effect of UGK17 on the growth of individual bacterial isolates. The UGK17 treatment reduced the CLas titers in both asymptomatic and symptomatic citrus leaves regardless of the age of the leaves. The UGK17 application augmented the PR gene expression. There is no statistically significant inhibition of bacterial growth or change of microbial community structure upon UGK17 treatment. Our results suggest that the chimeric peptide therapy is a promising solution to combat HLB by targeting gram-negative pathogens and enhancing the plant immune responses without impairing the beneficial microbial community in the citrus phyllosphere.

宿主源抗菌肽（host-derived antibacterial peptides）的应用已被视为防控柑橘最具毁灭性病害——黄龙病（Huanglongbing, HLB）的潜在高效安全手段。然而，引发HLB的病原菌如亚洲韧皮部杆菌候选种（Candidatus Liberibacter asiaticus, CLas）常会对宿主抗菌肽产生耐药性。本研究证实，包含两种不同宿主源抗菌肽的嵌合肽（chimeric peptides）不仅可保留抗菌活性，还能克服细菌耐药性并增强植物防御反应（plant defense responses）。此外，嵌合肽可能对植物相关微生物组（microbiome）的结构与功能产生脱靶效应（off-target impact）。但目前学界对于嵌合肽疗法在降低CLas滴度的同时，对柑橘叶围（phyllosphere）微生物群落结构的影响尚缺乏深入认知。本研究旨在评估嵌合肽UGK17（chimeric peptide UGK17）在降低CLas滴度、诱导植物防御反应以及影响柑橘叶围相关微生物组方面的应用效果。研究人员从美国德克萨斯州的橙树与葡萄柚树上采集叶片样本，依据成熟度将其划分为老叶与幼叶；同时根据感染状态与症状表现，将叶片分为三类：健康叶片、显症叶片（感染后表现典型症状）以及无症状叶片（感染后无明显症状）。体内实验采用将叶片分别浸没于0、5及25 μM的UGK17溶液中处理48小时的方法。通过实时定量PCR（quantitative real-time PCR, qPCR）与逆转录实时定量PCR（reverse transcription quantitative real-time PCR, RT-qPCR）分别完成CLas滴度与病程相关（PR）基因表达水平的定量检测；采用扩增子测序（amplicon sequencing）技术评估UGK17对细菌群落结构个体组成的影响。此外，本研究还开展了离体实验（ex planta assay），以探究UGK17对单一细菌分离株生长的影响。实验结果显示：无论叶片年龄如何，UGK17处理均可降低无症状与显症柑橘叶片中的CLas滴度；UGK17处理还能上调PR基因的表达。UGK17处理对细菌生长无显著抑制作用，也未引起微生物群落结构的显著变化。本研究结果表明，嵌合肽疗法是一种极具前景的HLB防控手段：其可靶向革兰氏阴性病原菌（gram-negative pathogens）并增强植物免疫反应，同时不会破坏柑橘叶围中的有益微生物群落。