Studies On Nematicidal Activity And Action Mechanism Of Virulence Factor PtxA In Pseudomonas Syringae

Plant Parasitic Nematodes?PPNs?are a variety of highly specialized omnivorous phytopathogenic nematode.They mainly inhabit the roots of host plants and can infect almost all crops,causing significant crop production loss by over 150 billion US dollars per year in the world.In the long-term practice of controlling agricultural pests,biological approaches,especially nematidical bacteria-based biocontrol technologies,are generally recognized as advantageous means to control PPNs infestation effectively,leads various naturally occurring or genetically modified nematicidal bacteria and their virulence substances drawing great attention worldwide in the recent years.Pseudomonas syringae MB03 is a wild-type strain that was isolated from the diseased plant tissue in this laboratory.Interestingly,previous work has confirmed that this isolate had nematicidal activity against not only the model nematode,Caenorhabditis elegans,but also the agriculturally important pest,Meloidogyne incognita.In this dissertation,a novel virulence factor,namely PtxA,which belongs to the hemolysin RTX family,was initially identified by sequence comparison.Its insecticidal activity,inhibition capabilities such as nematode growth,locomotion reduction and oviposition,as well as the feeding preference of worms,and its site of action in the worm body were examined,followed by functional characterization of structural domains and the possible receptors in worms.The main research results can be summarized as follows:Based on the genomic sequence of MB03,a prediction database of the virulence protein and virulence factors was initially constructed.The database shows that there are more than 200 potential direct or associated nematicidal enzymes or toxic proteins in MB03,which include the Rhs family,chitinases,collagenases,and hemolysin proteins,amongst others.Through further screening by the online tool,VirulentPred,a novel virulence factor PtxA of the hemolysin RTX family was identified.The PtxA gene from MB03 genome was cloned into Escherichia coli expression vector pET-28a,then was expressed and purified to obtain pure PtxA protein.The purified PtxA protein showed strong toxicity to C.elegans.The LC₅₀ value was 65.955?41.032¹25.246??g/mL,and the linear regression equation?correlation coefficient?can be defined as Y₁=-4.493+2.47 X₁.Meanwhile,this protein also exhibited high toxicity to M.incognita worms.The bioassay LC₅₀ was 139.825?30.139-264.375??g/mL,and the linear regression equation?correlation coefficient?can be defined as:Y₂=-6.259+2.917X₂.PtxA protein had a strong growth-inhibitory effect on C.elegans L1 worms.At a concentration of 315?g/mL,the length of the worms in the experimental group reached only 50%of the worms in the control group.In addition,PtxA protein can significant inhibit the number of eggs laid.The number of eggs laid by the nematode was significantly lower in the experimental group than in the control group under similar conditions.At the concentration of 252?g/mL,the average number of oviposites in the experimental group was only 30%of the control group.Feeding preference experiments also showed that C.elegans worms were more likely to feed on E.coli cells without expression of PtxA.Moreover,by using green fluorescence to label the intestine of the genetically modified C.elegans defective strain FT63,and using FT63 as a test worm,after 3 d of exposure to PtxA,tt was observed that the bamboo-like fluorescence structure of the experimental group was partially destroyed,strongly contrast to the complete structures of the control worms,indicating that the PtxA protein may cause physical damage to the intestinal tract of C.elegans worms.Furthermore,by using the pDS3.0 system to knock out the ptxA gene and comparing the toxicity of?PtxA and MB03 on PG plate,it showed that the C.elegans mortality caused by?PtxA strain plate was apparently less than that by MB03.To investigate whether two predicted domains of PtxA have different nematicidal activities or not,the corresponding coding fragment of these two domains were cloned into E.coli expression vector respectively,and the recombinant E.coli strains MB772 and MB773 were constructed,and the purified proteins DUF and M10_C were obtained after expression.A liquid nematicidal bioassay was conducted using C.elegans as the test worm.It showed that the two truncated structural domains were all toxic to worms,although the DUF protein was more toxic than M10_C.However,either DUF or M10_C was weaker than that of the full-length PtxA in term of toxicity,suggesting that the maximum toxicity of PtxA significantly depends on their combination and cooperation of both domains.Using PtxA as a bait protein,more than 20 potential receptor proteins were preliminarily identified from C.elegans by yeast two-hybrid assays.Real time quantitative PCR was performed to verify the expression of four receptor proteins from C.elegans,such as rps9,rps25,daf-21 and col-77.It showed that compared with the control group,the expression levels of these four genes were basically down-regulated after treating with PtxA for 12 h and 24 h,suggesting that PtxA's uptake affected the expression levels of these nematode genes.