The Virulence Exploring Of Transgenic Lecanicillium Attenuatum Against Citrus Whitefly (Diaelorude Citri) Mediated By RNA Interference

The citrus whitefly, Diaelorude citri (Asahmaed), is one of the most important citrus pests in the world. It feed on citrus leaf with their piercing-sucking mouthparts, causing massive fruit drop, more importantly, the nymph can secrete sticky honeydew, which can induce sooty mould, and finally undermine the photosynthesis. Currently, chemical control is the most effective strategies to control the citrus whitefly. However, the heavy use of the chemical agent has already caused negative problems, such as pesticide residues and insect resistance. Lecanicillium attenuatum (Ascomycetes, Hypocreales, Lecanicillium) is regarded as an important and fine entomopathogenic microorganism in pest biocontrol. The mycelium can cause the death of insect by penetrating the cuticle of the citrus whitefly and consuming its nutritio. With those irreplaceable advantages, L. attenuatum is regarded as an eco-friendly microbial pesticide. However, this agent is not widely used in the fields, due to its low virulence and long time infection. Fortunating, gene engineering technology was already conducted to balance this conflict by elevating the virulence in different methods. Thus, we constructed the RNA inteference vectors based on immune related genes of citrus whitefly and transformed plasmids into the L. attenuatum. We expected this method can enhance the virulence of entomopathogenic fungi with an idear as following: during the fungi infecting the whitefly, the dsRNA produced by ihpRNA expression cassette can be released from the fungi and interfere with target genes related with insect immunity, so reduced its immunity to fungi. The following is our results:1.Reconstruction of RNAi vector for filamentous fungiTo amplify PtrpC promotor and PtrpC+HygR component using the templates of pBHt2 plasmid; To amplify TtrpC terminator using the templates of pAN7-1 plasmid; Cut out 35s promotor of pFGC5941 plasmid using StuI and XhoI restriction enzymes and the remaining part was connected with PtrpC promotor on the corresponding site. The recombinant plasmid "PtrpC+Introrn" was finished. Cut out OCS terminator of "PtrpC+Intron" plasmid using SmaI and MssI restriction enzymes and the remaining part was connected with TtrpC terminator on the corresponding site. The recombinant plasmid "PtrpC+Intron+TtrpC" was finished. Cut out MAS+BlpR component of "PtrpC+Intron+TtrpC" plasmid using EcoRI and PstI restriction enzymes and the remaining part was connected with PtrpC+HygR component on the corresponding site. The recombinant plasmid "PtrpC+HygRPtrpC+Intron+TtrpC" was finished. Now the RNAi vector reconstruction was finished, we renamed this recombinant plasmid as pRCyl.2.Construction of RNAi vector based on immune related genes of citrus whiteflyFrom Diaelorude citri transcriptome database, we pay attention to three gene fragments related to its immune reaction, they are prophenoloxidase (PPO), prophenoloxidase activating factor (PPO-AF), lysozyme (LZM); Using the cDNA as the templates, we cloned the PPO, PPO-AF, LZM and PPO+PPO-AF fusion gene forward and reverse fragments succesfully; Connecting the forward fragments with pRCy1 vector on XhoI and SwaI restriction site, Connecting the reverse fragments with pRCy1 vector on XbaI and BamHI restriction site, then we finished the construction of ihpRNA expression cassette successfully.3.Agrobacterium-mediated genetic transformation of ihpRNA expression cas-sette into L. attenuatumThe ihpRNA expression vector was transformed into agrobacterium tumefacie-ns EHA105, then the ihpRNA expression cassette was integrated into the genom-e of L. attenuatum under the induction culture and co-culture treatment. Using selection medium which contain hygromycin B to verify the successfully trans-formed strains.4.Effect evaluation for transgenic L. attenuatumWe evaluated the unit sporulation and growth rate of transgenic fungi and found that the growth rate did not have significant change compared with wide type fungi; The unit sporulation also had no great different except La::GFP strain.The transgenic fungi were cultured for 10 generations continuously on PDA medium which had no hygromycin B, then assessed their genetic stability using specific primers,we found that all these fungi can be amplified specific bands, this shows that agrobacterium-mediated genetic transformation is a good way to ensure the transgene inherit stably in L. attenuatum.The citrus whitefly was treated with tansgentic fungi, then detected the expression level of interference target gene of whitefly using real-time quantitative PCR. We found that the L&::PPO strain, the La::PPO-AF strain and the La::LZM strain were all not have a significant silencing efficacy to their target genes compared with Lar::GFP strain. We speculated that this may because the dsRNA had a low expression level in L. attenuatum or it can not release into whitefly successfully.