Data_Sheet_4_Comparative Secretome and Functional Analyses Reveal Glycoside Hydrolase Family 30 and Cysteine Peptidase as Virulence Determinants in the Pinewood Nematode Bursaphelenchus xylophilus.ZIP

Pine wilt disease, caused by the pinewood nematode, Bursaphelenchus xylophilus, is one of the world’s most serious tree diseases. Although the B. xylophilus whole-genome sequence and comprehensive secretome profile have been determined over the past decade, it remains unclear what molecules are critical in pine wilt disease and govern B. xylophilus virulence in host pine trees. Here, a comparative secretome analysis among four isolates of B. xylophilus with distinct virulence levels was performed to identify virulence determinants. The four candidate virulence determinants of B. xylophilus highly secreted in virulent isolates included lipase (Bx-lip1), glycoside hydrolase family 30 (Bx-GH30), and two C1A family cysteine peptidases (Bx-CAT1 and Bx-CAT2). To validate the quantitative differences in the four potential virulence determinants among virulence groups at the protein level, we used real-time reverse-transcription polymerase chain reaction analysis to investigate these determinants at the transcript level at three time points: pre-inoculation, 3 days after inoculation (dai), and 7 dai into pine seedlings. The transcript levels of Bx-CAT1, Bx-CAT2, and Bx-GH30 were significantly higher in virulent isolates than in avirulent isolates at pre-inoculation and 3 dai. A subsequent leaf-disk assay based on transient overexpression in Nicotiana benthamiana revealed that the GH30 candidate virulent factor caused cell death in the plant. Furthermore, we demonstrated that Bx-CAT2 was involved in nutrient uptake for fungal feeding via soaking-mediated RNA interference. These findings indicate that the secreted proteins Bx-GH30 and Bx-CAT2 contribute to B. xylophilus virulence in host pine trees and may be involved in pine wilt disease.

由松材线虫（Bursaphelenchus xylophilus）引起的松材线虫病是全球最严重的林木病害之一。过去十年间，尽管学界已完成松材线虫全基因组测序并构建了全面的分泌组图谱，但目前仍不清楚哪些分子在松材线虫病中发挥关键调控作用，且调控松材线虫对宿主松树的毒力机制尚不明确。本研究针对4株毒力水平存在显著差异的松材线虫分离株开展比较分泌组分析，以筛选其毒力决定因子。在高毒力分离株中高分泌的4个候选松材线虫毒力决定因子分别为：脂肪酶（Bx-lip1）、糖苷水解酶家族30（Bx-GH30）以及2种C1A家族半胱氨酸肽酶（Bx-CAT1与Bx-CAT2）。为在蛋白水平验证4个潜在毒力决定因子在不同毒力组间的表达量差异，本研究采用实时反转录聚合酶链反应（real-time reverse-transcription polymerase chain reaction），在3个时间点（接种前、接种后3天（3 dai）及接种后7天（7 dai））对接种至松树幼苗的样本中这些因子进行转录水平检测。结果显示，在接种前及接种后3天时，高毒力分离株中Bx-CAT1、Bx-CAT2与Bx-GH30的转录水平显著高于无毒力分离株。后续基于本氏烟草（Nicotiana benthamiana）瞬时过表达的叶盘实验表明，候选毒力因子GH30可诱导植物产生细胞死亡。此外，本研究通过浸泡介导的RNA干扰（RNA interference）证实，Bx-CAT2参与了真菌取食所需的营养摄取过程。上述研究结果表明，分泌蛋白Bx-GH30与Bx-CAT2可调控松材线虫对宿主松树的毒力，可能参与松材线虫病的致病过程。