Department of Chemical and Environmental Engineering, University of California Riverside, Riverside, California, USA.
Department of Microbiology and Plant Pathology, University of California Riverside, Revierside, California, USA.
Biotechnol Bioeng. 2021 Aug;118(8):3200-3214. doi: 10.1002/bit.27845. Epub 2021 Jun 6.
In plants, polygalacturonase-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromises plant cell walls and leaves the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1 and BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. We found that tPvPGIP2_5-8, which contains LRR5 to LRR8 and is only one-third the size of the full length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full length PvPGIP2 via Y2H. The inhibitory activities of tPvPGIP2_5-8 on the growth of A. niger and B. cinerea were then examined and confirmed on pectin agar. On pectin assays, application of both full length PvPGIP2 and tPvPGIP2_5-8 clearly slows down the growth of A. niger and B. cinerea. Investigation on the sequence-function relationships of PGIP utilizing a combination of site directed mutagenesis and a variety of peptide truncations suggests that LRR5 could have the most essential structural feature for the inhibitory activities, and may be a possible target for the future engineering of PGIP with enhanced activity. This study highlights the potential of plant-derived PGIPs as a candidate for future in planta evaluation as a pest control agent.
在植物中，多聚半乳糖醛酸酶抑制蛋白（PGIPs）通过抑制果胶解聚酶（PGs）的多聚半乳糖醛酸酶活性，在抵抗真菌疾病方面发挥着关键作用。PGs 是一种由病原体分泌的酶，会破坏植物细胞壁，使植物易受疾病侵害。在这里，通过酵母双杂交（Y2H）系统重建了菜豆（PvPGIP1 和 PvPGIP2）和黑曲霉（AnPG2）、灰葡萄孢（BcPG1 和 BcPG2）以及串珠镰刀菌（FmPG3）PGs 之间的相互作用，以研究各种 PvPGIP1 和 2 截断和突变体的抑制效率。我们发现，包含 LRR5 到 LRR8 的 tPvPGIP2_5-8 仅为全长肽的三分之一大小，但通过 Y2H 与全长 PvPGIP2 与 AnPG 和 BcPG 具有相同的相互作用水平。然后，我们通过果胶琼脂实验检测和证实了 tPvPGIP2_5-8 对黑曲霉和灰葡萄孢生长的抑制活性。在果胶实验中，全长 PvPGIP2 和 tPvPGIP2_5-8 的应用都明显减缓了黑曲霉和灰葡萄孢的生长。利用定点突变和多种肽截断的方法对 PGIP 的序列-功能关系进行研究表明，LRR5 可能具有抑制活性的最基本结构特征，并且可能是未来具有增强活性的 PGIP 工程的潜在目标。这项研究强调了植物来源的 PGIPs 作为未来作为害虫防治剂在植物体内评估的候选物的潜力。
