In vitro uptake of 140 kDa Bacillus thuringiensis nematicidal crystal proteins by the second stage juvenile of Meloidogyne hapla

Plant-parasitic nematodes (PPNs) are piercing/sucking pests, which cause severe damage to crops worldwide, and are difficult to control. The cyst and root-knot nematodes (RKN) are sedentary endoparasites that develop specialized multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of the Meloidogyne hapla. Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla. To monitor the entry pathway of crystal proteins into M. hapla, second-stage juveniles (J2) were treated with NHS-rhodamine labeled nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba). Confocal microscopic observation showed that these crystal proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the stylet, and it has important implications in designing a transgenic resistance approach to control RKN.