Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technologygrid.6357.7, Nakhon Ratchasima, Thailand.
Biofertilizer & Plant-Microbe Interaction Research Unit, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technologygrid.6357.7, Nakhon Ratchasima, Thailand.
Microbiol Spectr. 2021 Dec 22;9(3):e0209421. doi: 10.1128/Spectrum.02094-21. Epub 2021 Dec 15.
is an endophytic bacterium under investigation as an efficient biofertilizer for sustainable legume-rice rotational cropping system. Monitoring and bio-imaging of this nitrogen fixing bacterium is essential for the study of plant-microbe evolution, soil microbiome, as well as quality control in organic farming. While phage display antibody technology has been widely used to generate recombinant antibody for myriad medical purposes, so far, this technology has been minimally applied in the agricultural sector. In this study, single-chain variable fragments (scFv) against two strains SUTN9-2 (yiN92-1e10) and DOA9 (yiDOA9-162) were isolated from a human phage display antibody library. Specific binding of scFv was demonstrated by ELISA and confocal-immunofluorescence imaging techniques. localization in both endophytic and bacteroid forms could be observed inside rice tissue and plant nodule, respectively. Moreover, successful application of the recombinant antibody for the evaluation of nodule occupancy was also demonstrated in comparison with standard GUS-staining method. The results of this study showed for the first time the potential use of human phage display scFv antibody for imaging and monitoring of biofertilizer and thus could be further applied for point-of-detection of bacterial inoculum in the legume-rice rotational crop system. Human scFv antibody generated from phage display technology was successfully used for the generation of specific recombinant antibodies: yiN92-1e10 and yiDOA9-162 for the detection of strains SUTN9-2 and DOA9, respectively. These two recombinant scFv antibodies could be used for precise detection of the rhizobia both in symbiosis with legume and endophyte in rice tissue by ELISA and immunofluorescent staining, during legume-rice rotational cropping system in the field. This methodology can be further employed for the study of other plant-microbe interactions and monitoring of biofertilizer in diverse sustainable cropping systems as well as in precision agriculture.
是一种内生细菌，正在作为可持续豆科-水稻轮作系统的高效生物肥料进行研究。对这种固氮细菌的监测和生物成像对于研究植物-微生物进化、土壤微生物组以及有机农业中的质量控制至关重要。虽然噬菌体展示抗体技术已广泛用于为众多医疗目的生成重组抗体，但迄今为止，该技术在农业领域的应用很少。在这项研究中，从人类噬菌体展示抗体文库中分离出针对两个菌株 SUTN9-2（yiN92-1e10）和 DOA9（yiDOA9-162）的单链可变片段（scFv）。通过 ELISA 和共聚焦免疫荧光成像技术证明了 scFv 的特异性结合。在水稻组织和植物根瘤中分别观察到 scFv 在内生和菌体型中的定位。此外，还通过与标准 GUS 染色方法的比较，成功地将重组抗体应用于评估根瘤占有率。这项研究的结果首次表明，噬菌体展示 scFv 抗体可用于生物肥料的成像和监测，因此可进一步应用于豆科-水稻轮作系统中细菌接种剂的现场检测。噬菌体展示技术产生的人源 scFv 抗体成功用于生成特异性重组抗体：yiN92-1e10 和 yiDOA9-162，分别用于检测 SUTN9-2 和 DOA9 菌株。这两种重组 scFv 抗体可用于通过 ELISA 和免疫荧光染色在田间豆科-水稻轮作系统中精确检测与豆科植物共生和内生在水稻组织中的根瘤菌。该方法可进一步用于研究其他植物-微生物相互作用以及监测不同可持续种植系统和精准农业中的生物肥料。
