Table 1.
The information of Cry55Aa, Cry6Aa, and Cry5Ba used in this study.
Figure 1.
The dose response curves of Cry6Aa, Cry55Aa, and Cry5Ba against M. hapla J2 in the presence of resorcinol (RES) or tomato root exudates (TRE).
The bioassay of three nematicidal crystal protein Cry6Aa (A), Cry55Aa (B), Cry5Ba (C) against M. hapla J2 were conducted in the presence of resorcinol (RES), or tomato root exudates (TRE), or ddH2O (CK), respectively. A non-nematicidal crystal protein Cry1Ac (D) was treated as the same and used as control. The M. hapla J2 were exposed to five doses of each crystal proteins. Data shown represent the percentage of animals that were intoxicated when fed crystal proteins. Error bars represent the S.D. from the mean of averages over three independent experiments. Each data point represents the average size of 60 animals. The mortality was 3.3% in the absence of any toxins.
Figure 2.
Activities of rhodamine labeled Cry6Aa, Cry55Aa, and Cry5Ba against M. hapla J2.
M. hapla J2 were exposed to three doses of non-labeled crystal protein or rhodamine labeled Cry6Aa (A), Cry55Aa (B), Cry5Ba (C), and rhodamine-6G (D) in the presence of resorcinol. M. hapla J2 were exposed to three doses of crystal protein. Data shown represent the percentage of animals that were intoxicated when fed crystal proteins or rhodamine labeled crystal protein. Each data point represents the average size of 60 animals. Error bars represent the S.D. from the mean of averages over three independent experiments.
Figure 3.
The pathway of nematicidal crystal proteins entering M. hapla J2.
The confocal laser scanning microscope image showed the ingestion manner and process of Cry55Aa (A), Cry6Aa (B), or Cry5Ba (C) by M. hapla J2 in the presence of resorcinol (Res) or tomato root exudates (TRE). M. hapla J2 were incubated in rhodamine-labeled crystal toxins for three different times then imaged using a merged image. The rhodamine 6G (D) was treated as the same and used as a control. Toxin was detected inside the treated M. hapla J2, but not in the control (CK). The anterior of M. hapla is positioned within the upper region. Abbreviation: s = stylet; el = esophageal lumen; h = head of M. hapla J2. The scale bar of all the images is 40.43 µm.
Figure 4.
Detection of size changes of Cry55Aa, Cry6Aa, and Cry5Ba in M. hapla J2 by Western blot analysis.
M. hapla J2 were incubated with Cry55Aa protein (Panel A) and Cry6Aa protein (Panel B), and then detected by Western blot at 0, 12, 36, and 72 hpi, using an anti-crystal antibody; M. hapla J2 were incubated with Cry5Ba protein (Panel D, F) and then detected by Western blot at 0, 12, 22, 50, and 96 hpi, using Cry5Ba protein antibody. Line CK: Controls of crystal protein without being incubated by M. hapla J2. Panel C: SDS-PAGE showed the molecular mass of purified Cry5Ba. Panel E: the Cry5Ba was incubated by M. hapla J2 for 12 h, centrifuged at 12000 rpm for 10 min to remove M. hapla J2, and then detected by Western blot using Cry5Ba antibody.
Figure 5.
Enzyme-linked immunosorbent assay analysis of the uptake efficiency of nematicidal crystal proteins by M. hapla J2.
The uptake efficiency was determined by subtracting the percentage crystal proteins uptake in the absence of resorcinol from that in the presence of resorcinol. Each bar value represents the mean SD of triplicate experiments.