Nematicidal Mechanisms Of Bacillus Thuringiensis Cry6A And Cry5B Proteins

Plant-parasitic nematodes are widely distributed,with a large number of species,causing enormous economic losses to agricultural production.Up to now,there are many methods to control plant parasitic nematodes,among which the biocontrol has the characteristics of high efficiency and safety,and has attracted more and more attention.Bacillus thuringiensis(Bt)represents an important biocontrol bacterium,which can control not only insect pests,but also plant-and animal-parasitic nematodes.Bt has a variety of toxins and virulence factors against nematodes,including proteins and small molecules,among which Cry proteins are the most concerned,mainly classified as Cry5 family and Cry6 family.At present,the research on nematicidal mechanisms of Cry proteins is very limited,mainly including the following progress: Cry6 A can induce the death of Caenorhabditis elegans through the necrosis signalling pathway;Cry5B binds to the glycolipids of C.elegans and forms pores in the intestinal cell membrane,leading to the death of nematodes.Cry6 A receptors and intracellular mechanisms of Cry5 B have not yet been reported.Therefore,this study focuses on two unknown areas about the nematocidal mechanism of Cry proteins.C.elegans,a free-living nematode,has simple genetic manipulation and high homology with plant-parasitic nematodes.C.elegans has a strong guiding role in the study of plant-parasitic nematodes.In order to carry out the project better,this study chose C.elegans as the target nematode of Cry proteins.By C.elegans-Cry proteins system,the nematocidal mechanism of Cry6 A and Cry5 B was investigated in this study.The contents of this study are as follows:1.The GPI-anchored protein RBT-1 of C.elegans is a functional receptor of Cry6 A.Firstly,RBT-1 can interact with Cry6 A.The reported Cry protein receptors are mainly GPI-anchored proteins.Therefore,the screening range of Cry6 A receptors was selected for the GPI-anchored proteins of C.elegans.By screening the GPI-anchored proteins from C.elegans using affinity chromatography,this study found a Cry6A-interacting protein,F35E12.10(called RBT-1).RBT-1 is a GPI-anchored protein located on the lipid raft on the intestinal cells of C.elegans,containing an N-terminal signal peptide and glycosylation sites.Western blot and ELISA further demonstrated the interaction between RBT-1 and Cry6 A,and the dissociation constant was about 21.5 n M.Secondly,RBT-1 is involved in the nematicidal process of Cry6 A.Through tracking experiments of rhodamine-labeled Cry6 A,we found that RBT-1 could also help this Cry protein interact with the intestinal cells of C.elegans.The results of tracing with propidium iodide showed that RBT-1 was involved in the perforation of Cry6 A in gut cells of nematodes.Bioassay results show that rbt-1 mutation can cause the resistance of nematodes to Cry6 A,suggesting that RBT-1 could mediate the nematocidal activity of Cry6 A.These results indicate that RBT-1 is a specific receptor of Cry6 A and represents a new class of Cry protein receptors.2.Mitochondria are the intracellular nematicidal targets of Cry5 B in C.elegans.At present,there is no report on the intracellular nematocidal events of Cry5 B.The purpose of this study is to reveal the intracellular nematocidal mechanisms of Cry5 B.By means of cell biology,molecular biology and genetics,we found that mitochondria were important insecticidal targets of Cry5 B.Firstly,Cry5 B inhibited the activity of mitochondrial respiratory chain complex I and further reduced the mitochondrial membrane potential of nematodes,which was involved in the nematocidal process of Cry5 B.Secondly,Cry5 B caused the decrease of mitochondrial ROS.Bioassay results showed that ROS enhenced the nematocidal activity of Cry5 B.Thirdly,Cry5 B also caused changes of mitochondrial morphology,which was caused by the decrease in mitochondrial membrane potential and was involved in the nematocidal process of Cry5 B.Fourthly,it was also found that the drop of mitochondrial membrane potential induced by Cry5 B also resulted in the decrease of mitochondrial content,which was also involved in the nematocidal process of Cry5 B.Finally,systemic damage induced by localized Cry5 B attack represents a novel pattern of pathogenic effect.Therefore,mitochondria are Cry5B-sensitive organelles,and are the important intracellular nematicidal targets for Cry5 B.This finding represents a novel working mechanism of Cry proteins.