Targeting Of Anti-microbial Proteins To The Hyphal Surface And Nanochitin Suspension Amplifies Protection Of Crop Plants Against Phytophthora Pathogens

The genus of Phytophthora belongs to oomycetes from the kingdom Stramenopila,and consists of nearly 200 described species,including Phytophthora infestans,the causal agent of potato late blight;P.sojae,causing ‘damping off’ of seedlings and root rot of soybean;and P.capsici,causing Pepper blight.The genus continues to be a threat to global food,vegetables and forestry production security.The management of Phytophthora diseases is an ongoing challenge despite improvements in chemical pesticides and resistance breeding because the pathogens have proved to be highly diverse in terms of their lifestyles,high pathogenicity,highly adaptable,remarkabe adaptive to overcome pesticides and resistant genes,which cause highly risk of disaster.Hence,it’s urgent to find broad spectrum and durable strategies to control Phytophthora diseases,which is the focus of researchers in genetic breeding and plant phytopathologist.Our previous studies showed that Phytophthora sysnthesis PI3 P to bind to RXLR effectors,and aid to its translocation and stabilization to improve virulence during infection,which showed that PI3 P is the indispensable weapon.Hence,we thought that PI3 P is a potential target for accurate design in breeding for disease resistance.The fusion of PI3 P specific binding peptide(FYVE)and antimicrobial protein was constructed to obtain transgenic plants.The results showed that FYVE could guide AMPs to the surface of Phytophthora,furtherly enhance the AMPs activity to Phytophthora to improve the plant resistance.We proposed a new strategy for Phytophthora control: targeting of anti-microbial proteins to the hyphal surface amplifies protection of crop plants against Phytophthora pathogens.Meanwhile,we found that the new bio-nanomaterials called nanochitin improved plant resistance to Phytophthora depended on CERK1 and BAK1.The main results and conclusions are shown as follows:Targeting of anti-microbial proteins to the hyphal surface amplifies protection of crop plants against Phytophthora pathogens.Using tobacco transient expression system mediated by Agrobacterium tumefaciens,the anti-Phytophthora activity of Lactoferrin(Lac),Nm Def02,GAFP1 and GAFP3 were test.The results showed that the leaves expressing GAFP1 or GAFP3 exhibited significant colonization reduction by two pathogens,P.capsici and P.parasitica.Then,a set of constructs encoding a tandem repeat of the EEA1 FYVE domain(2x FYVE)fused with GAFP1 or GAFP3(SP-GFP-GAFP1/3-FYVE)were generated and expressed in N.benthamiana leaves,and we found that the resistance conferred by both proteins was significantly strengthened by fusion with the 2x FYVE domain.To analyse the mechanism of SP-GAFP1/3-FYVE,the subcellular of SP-GAFP1/3-FYVE was observed,results showed that GAFP1/3-FYVE could accumulate around the haustoria and invasive hyphae,in contrast,the GAFP1/3 could not accumulate around pathogen hyphae,these results confirm that the 2x FYVE domain is effective in increasing the targeting of GAFP1 and GAFP3 to the pathogen to improve plant resistance.Meanwhile,we determined the in-vitro activity of GAFP1/3-FYVE,and found that GAFP1/3-FYVE fusion significantly inhibit germ tube elongation of germinating zoospores and virulence of Phytophthora.Together,expressing GAFP1/3-FYVE in plant could significantly improve plant resistance to Phytophthora,which caused by 2x FYVE domain targeting the anti-microbial proteins to accumulate on the Phytophthora hyphae.Our previous study showed that there were five predicted PI3 K genes were related to PI3 P content.We generated knock-out strains in P.sojae strain P6497 using CRISPR-Cas9 technology,and found that PI3K1/2/5 are important for pathogenicity.By lipid binding assay and PI3 K enzyme activity assay,results showed that PI3K1 and PI3K2 proteins,but not PI3K5,could bind to PI3 P.Intrestingly,we found the fluorescence signals of GFP-FYVE were substantially reduced in the pi3k1-deletion and pi3k2-deficiency mutants,but not pi3k5,which indicating that PI3K1 and PI3K2 are responsible for the stainable PI3 P on the surface of the hyphae.To determine the effect of the pi3 k mutations on infection of soybean lines expressing GAFP-FYVE fusions,we made mutations in a second P.sojae strain,P7076,which was unaffected by the Rps1 k resistance gene in the soybean lines.We used confocal microscopy to examine the invasive hyphae of P.sojae on etiolated seedlings of the transgenic soybean line expressing SP-GFP-FYVE.We observed that fluorescent signals of SP-GFP-FYVE specifically accumulate around the hyphae of P7076 and Ctrl,but this accumulation is absent with the pi3 k mutants.Together,these results demonstrate that PI3K1 and PI3K2 are essential for function of FYVE domain.NC improved plant resistance dependent on CERK1 and BAK1.We use model of interaction system of plant and Phytophthora to explore the mechanism of NC,and the results showed that NC did not exhibit inhibitory effect on vegetative growth of P.capsici,but enhanced the resistance against P.capsici by PR gene expression.Furtherly,P.capsici resistance,PTI maker gene promotion,and ROS production in A.thaliana induced by NC depended not only on chitin receptor CERK1,but also BAK1.And Flg22 induced resistance to P.capsici in Arabidopsis also depends on BAK1 and CERK1.Hence,NC,as a MAMP,induced plant resistance through a mechanism of a crossmicrobe protection via the BAK1-CERK1 pathway in plant,pointing to the complexity of the plant immunitysystem.Overall,we found that PI3 P content of Phytophthora is closely related to the virulence,which indicated PI3 P could act as target for prevention.Using PI3 P specific binding peptide(FYVE)guide GAFP1/3 accumulating to the Phytophthora significantly improves plant resistance against Phytophthora pathogens.We showed that this strategy can provide stable transgenic soybean and potato lines with improved resistance against P.sojae and P.infestans respectively,without any negative effects on growth or yield.Hence,we have successfully proposed and verified a molecular breeding approach to improve plant resistance to Phytophthora,and provided potential transgenic plant with high resistance,and also establish the foundation of controlling other crop pathogens.Meanwhile,we also analyse the molecular mechanism of systemic acquired resistance of NC,and provided a theoretical basis for the application of nanochitin in agriculture.