Isolation And Identification Of Pratylenchus Species And Functional Study Of Effector Proteins Pc-CZ、Pc-CD

Pratylenchus spp.are also known as root-lesion nematodes,is a kind of important migratory plant parasitic nematodes.Root-lesion nematodes are widely distributed in the world and have a wide range of host.Root-lesion nematodes and root-knot nematode and cyst nematode is the most severe damage on the crops of three kinds of plant parasitic nematodes.Because of its small size and the hidden nature of infection,the harmful of root lesion nematodes in agricultural production is often neglected.Pratylenchus coffeae is one of the most serious root-lesion nematodes in our country,which cause serious damage to food crops and cash crops.P.coffeae can cause of crop root-rot disease,and has become a kind of the important diseases on agricultural production in China,also cause a serious threat to agricultural production safety,due to its strong colonization,the nematode control still lacks effective methods.At present,the study of P.coffeae effector proteins are very scarce,and isolating and identifying the effector proteins expressed by the esophagus glands of Pratylenchus and clarifying the function of these effector proteins are the key to understanding the molecular mechanism of P.coffeae infection and its interaction with hosts.In this paper,9 populations of nematodes were isolated and identified through morphological and molecular biology methods,and 3 species of Pratylenchus and 1 species of Helicotylenchus were obtained.The highly pathogenic population of P.coffeae SY-1 was purified and cultured,and the full-length sequences of the cathepsin Z and D genes(Pc-CZ and Pc-CD)of this nematode were obtained.In this study,the expression characteristics of Pc-CZ and Pc-CD in P.coffeae are explored by a variety of molecular techniques.RNAi and transient expression were used to verify the function of these effector protein in the pathogenic process of P.coffeae infection and its influence on host defense response.The interaction proteins of PC-CZ in the maize library were preliminarily screened by yeast double hybridization.The main results are obtained as follows:1.Isolation and identification of nematodesThis study uses the Berman funnel method to isolate the nematodes in the samples collected from various cities,and the species identification is carried out by integrative morphological and molecular analyses.The identification results showed that the 9 different populations of nematodes included five populations of P.scribnei,two populations of P.coffeae,one population of P.neglectus and one population of H.microlobus,of which the P.coffeae A-21 population was first reported on Chinese sesame seeds,the P.scribnei SC-1 population and HN-1 population respectively were the first report on tomatoes in Sichuan Province and Maize in Hainan Province,the P.neglectus SY-X population was the first report in Maize Henan Province,and H.microlobus LQ-1 population was first report in Henan Province.In addition,the eight populations of these three Pratylenchus were purified and propagated on carrot callus.2.Cloning and sequence analysis of effector protein Pc-CZ and Pc-CD in P.coffeae.The full-length c DNA sequences of Pc-CZ and Pc-CD genes were obtained by race technology.The full-length of Pc-CZ gene is 1619 bp,ORF 1461 bp,encoding 486 aa,and there is a 26 aa signal peptide at the N-terminal.The theoretical molecular weight(MW)of the protein is 51.2 k Da and the Isopotential point is 7.46;The full-length of Pc-CD gene is 1698 bp,ORF 1446 bp,encoding 481 aa.There is a 24 aa signal peptide at the N-terminal.The theoretical molecular weight(MW)of the protein is 53.2 k Da and the Isopotential point is 5.52.3.Expression characteristics and tissue localization of effector protein genes Pc-CZ and Pc-CD of P.coffeae.q RT-PCR was used to detect the expression levels of Pc-CZ and Pc-CD genes in four different stages of P.coffeae(female,male,larva and egg).The results showed that Pc-CZ and Pc-CD genes were expressed in all four stages of P.coffeae,and the highest expression levels were found in females.The expression level of Pc-CZ was the lowest in males,and that of Pc-CD was the lowest in eggs.Both Pc-CZ and Pc-CD were specifically expressed in the esophageal glands of P.coffeae by in situ hybridization.4.Subcellular localization and Western blot detection of effector proteins Pc-CZ and Pc-CD of P.coffeae.The recombinant proteins Pc-CZ and Pc-CD were expressed in N.benthamiana leaves by agrobacterium-mediated method,and their localization in plant cells was analyzed.Subcellular localization showed that Pc-CZ and Pc-CD could detect fluorescence signal of target protein in cytoplasm and nucleus of N.benthamiana.5.Function of effector protein genes Pc-CZ and Pc-CD in the pathogenic and reproductive process of P.coffeae infection.In vitro ds RNA silencing method was used to confirm that the silencing efficiency of target genes Pc-CZ and Pc-CD increased with the extension of treatment time in a certain time range.When Pc-CZ was treated with RNAi for 24 h,the expression of Pc-CZ decreased by 78% compared with the control group,and the silencing efficiency was highest at this time;The silencing efficiency of Pc-CD was the highest after 12 h RNAi treatment,and the expression of Pc-CD decreased by 81.4 % compared with the control group.After treatment with Pc-CZ gene RNAi for 24 h,the fecundity of P.coffeae on carrot callus decreased by 72.5% compared with the control group,and the pathogenicity of P.coffeae to tomato was also significantly reduced in the treatment group.After 12 h treatment with Pc-CD gene RNAi,the fecundity of P.coffeae on carrot callus decreased by 66.5 % compared with the control group,and the virulence of P.coffeae to tomato was also significantly reduced.After 48 h of fuchsia staining,the number of nematodes in tomato roots treated with Pc-CZ and Pc-CD gene RNAi for 24 h and 12 h was significantly lower than that of the control group,indicating that RNAi treatment significantly reduced the infectivity of P.coffeae to the host.These results suggest that effector protein genes Pc-CZ and Pc-CD play an important role in the pathogenic and reproductive process of P.coffeae infection.6.Effects of effector proteins Pc-CD and Pc-CZ on host defense responseTransient expression of Pc-CZ and Pc-CD proteins in N.benthamiana leaves by Agrobacterium mediated method could significantly inhibit the expression of PTI related genes Pti5,Grans2 and Acre3,and down regulated the transcription levels of PAL and LOX in salicylic and jasmonic acid pathways in N.benthamiana leaves.Transient expression of Pc-CD and Pc-NS-CD in N.benthamiana leaves significantly inhibited the transcription levels of LOX and PR1 in salicylic acid and jasmonic acid pathway compared with control e GFP.The expression of PTI-related genes Pti5,Grans2 and Acre3 in Pc-CD gene was not significantly down-regulated compared with control e GFP,while the expression of PTI-related genes Grans2 and Acre3 in Pc-NS-CD gene was significantly inhibited compared with control e GFP.Both cathepsin Z and D of P.coffeae can significantly inhibit the flg22 induced outbreak of reactive oxygen(ROS).In addition,they also inhibit Rbp1/Gpa2 mediated programmed cell death.7.Verification of PC-CZ interacting proteins in Maize library by yeast two-hybridRecombinant protein PGBKT7-Pc-CZ was non-toxic to yeast cells and had no self-activation activity.Through yeast two hybrid library screening technology,it was preliminarily confirmed that 8 proteins in Maize library interacted with Pc-CZ protein of P.coffeae.