Study On The Proteolytic Properties Of Xnpt Toxin Complexes From Xenorhabdus Nematophila

Xenorhabdus nematophila HB310 (Xn HB310), screened in our laboratory, is insect pathogen bacteria symbiotically associated with entomopathogenic nematode Steinernema carpocapsae. Xn HB310 had high and broad spectrum insecticidal activity, and produces the insecticidal toxin complex proteins(Xnpt). Two Xnpts ( Xnpt1 and Xnpt2) were purified from the intracellular protein of Xn HB310 by native-PAGE in the preliminary research. The Xnpt1 and Xnpt2 all are composed of three different proteins (Xnpt1:XptA1, XptB1 and XptC1; Xnpt2: XptA2, XptB1 and XptC1). Xnpt1 is involved in activity towards Pieris rapae, while the Xnpt2 is responsible for activity towards Helicoverpa armigera and Plutella xylostella. In order to clarify the insecticidal mechanism of Xnpt toxin, the proteolytic properties of Xnpt toxins were studied by in vitro digestion of the insect midgut proteinase and trypsin. In addition, the interactive resistance and the synergistic action between Xn HB310 and Bacillus thuringiensis (Bt) HD-73 were determined in the laboratory. The results were as the following.1. After treated in vitro by larvae midgut juinces from P. rapae, H. armigera, Mythimna separata, Hyphantria cunea and Prodenia litura, main proteins of Xnpt and Xnpt2 all were degraded to become two new proteins band of 113 kDa and 60 kDa. In addition, Xnpt toxins and Xnpt2 toxin could also were cleaved by trypsin into the same two new protein fragments of 113 kDa and 60 kDa. However, they could not be digested by pepsin and proteinase K.Two new protein bands of 113 kDa and 60 kDa were identified as XptA2 protein fragments by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS). The results suggested that it may be trypsin-like in insect midgut which was key proteases for Xnpt2 proteolytic processing.2. After treated Xnpt toxin with different midgut juices or trypsin at 24 h, the insecticidal activities of Xnpt toxin to larvae of H. armigera and P. xylostella did not happen obvious change. The results suggested that the proteolytic properties of Xpnt2 toxin in insect midgut may be similar to that of Bt Cry toxins.3. By means of resistant-selecting with Bt HD-73 in the laboratory. one Bt- resistant diamondback moth populations (DBM-R) was gotten. DBM-R had 105.43-fold resistance to Bt HD-73 and 30.70-fold resistance to Bt HD-1. The LC50s of the culture broth from Xn HB310 to Bt-susceptible and Bt-resistant populations of P. xylostella were measured by the method of leaves-tip bioassays. The results showed that Bt-resistant population of P. xylostella just only had 0.92-fold resistance to Xn HB310. It demonstrated that there was not interactive resistance between Xn HB310 and Bt HD-73. After assaying the synergistic effect between Xn HB310 and Bt HD-73, it was found that Bt HD-73 had significant synergistic effect on Xn HB310 against the 3rd instar larvae of P. xylostella. However, Xn HB310 had not the synergistic effect on Bt HD-73.