Effector Proteins Secreted By Phytophthora Infestans Under Nitrogen Starvation

The late blight of potato caused by Phytophthora infestans（Mont.）de Bary has been regarded as one of the most serious fungal diseases affecting the potato production.Late blight caused by P.infestans causes annual losses（costs of control and damage）estimated at more than €1,000,000,000 in the world.It has been shown that nitrogen starvation was the main environmental stress factor encountered in the early stage of pathogen invasion.Nitrogen deficiency can cause specific expression of pathogenesis related genes of the fungus.The nitrogen-deficient environment faced by the pathogens at the early stage in the host plants can be simulated by in vitronitrogen starvation liquid culture conditions.When the pathogen infects the potato plant（Solanum tuberosum L.）,it can secrete effector proteins with Rx LR motif which were found to be high in expression in the early stage of pathogen infection.These proteins combine with particular sites of the host plant and change the defense mechanism of the host.It further helps the pathogen to make complete colonization in the cells,eventually leading to the loss of resistance of the host plant.Therefore,it is a matter of great significance that by exploring in deepth study of these effectors with Rx LR motif the characteristics of early pathogen infection could be revealed.We obtained one P.infestans strain（NOD-1）which can secrete proteins that cause obvious necrotic spots on potato leaves under nitrogen（N）starvation conditions.The effect,thus indicates that NOD-1 produces strong pathogenesis associated proteins under nitrogen-deficient culture condition of potato plant.However,the nature of these proteins,as well as whether they contain secreted effectors with the Rx LR motif have not yet been elucidated properly.In this thesis,NOD-1,a strain of late blight of potato disease screened in the laboratory has been used as the research material.The pathogenicity of the secreted protein produced by this strain was analysed by using a model plant（Nicotiana benthamiana Domin）.Research results from the present thesis showed that NOD-1 produced the pathogenic secreted protein when cultured for 12 d and 22 d,separately under N starvation stress.The secreted proteins of NOD-1 were analysed using high throughput liquid chromatography-tandem mass spectrometry（LC/MS/MS）of the tag（Label-free）quantitative proteomic techniques.The analysis reveals the presence of 3 effect proteins with Rx LR motif.The two secretory proteins namely,DONTI4 and DOMQL8 obtained from the prokaryotic expression and purification had proved to be active in the host plant.Pathogenic analysis carried out showed that the two proteins were pathogenic disease related effects secretion proteins.The main results of the present thesis have been summarised as follows.1.Secretory effector protein of NOD-1 induced by N starvationThe mycelia of the P.infestans NOD-1 strain was transferred to a complete liquid medium as well as in N starvation liquid medium separately,and incubated in a shaker at 17 ℃and 150 rpm for 24 days.The secreted proteins were extracted and purified every 3rd day from the beginning of the experiment.They were inoculated into N.benthamiana plant with 1.0 μg/μl concentration.At the 3rd day（72 h）of inoculation,the necrosis in N.benthamiana was detected as water-soaked spots for the strain cultivated for 12 d and 22 d under N starvation and 16 d in a complete medium,respectively.However,the necrotic spot area due to the inoculation of secreted protein isolated from 22 d N starvation condition was almost twice as strong as that of the 16 d complete culture.The results had indicated two sequences.In one hand,the nitrogen-deficient environment could induce high expressions of pathogenesis-related genes,maybe including effector genes.On the other hand,there was a satisfactory differentiation and stability that the pathogenicity of secreted protein of the P.infestans NOD-1 strain was identified by N.benthamiana.As well as the culture time for producing pathogen proteins secreted under nitrogen starvation was also proved to be definite.2.Screening for secretory effector proteins produced by NOD-1 strain under N starvation culture using Label-free LC-MS methodNOD-1 strain was cultured in the complete nutrition and N starvation liquid media for 12 d and 22 d.The secreted proteins of NOD-1 were identified using high throughput liquid chromatography-tandem mass spectrometry（LC/MS/MS）of the tag（Label-free）quantitative proteomic techniques.According to the identification results,the secreted differentially expressed proteins were analyzed by bioinformatics softwares,and the results showed that the strain in Tq N22（N starvation cultured for 22d）yielded 146 secreted proteins（including co-differentially expressed and specifically expressed,the same below）and the Tq N12（N starvation cultured for 12d）showed 17 differentially expressed proteins.On the basis of the number of differentially expressed secreted proteins,the former was 8.59 times higher than the latter.However,by the CW22（full nutrient cultured for 22d）and CW12（full nutrient cultured for 12d）about 96 and 26 differentially expressed proteins were obtained,respectively.In this case the former is3.69 times higher than the latter.Comparing the results of the two treatments with the same culture time,it was found that the differentially expressed proteins obtained after CW12 treatment was 1.53 times greater than that of the Tq N12.As a whole,the differentially expressed proteins in Tq N22 was 1.52 times greater than that of the CW22.These results showed that the amount of differential proteins secreted by NOD-1 was higher at 22 d than at 12 d and the increase was greater in N starvation culture.In addition,the number of differential proteins secreted under the condition of Tq N12 was less than that of CW12.But at 22 d,Tq N22 produced significantly more secreted proteins than that of CW22.The analysis of the results of the effector proteins obtained from NOD-1 strains under the two culture conditions showed the presence of common effector proteins expressed in Tq N12 and CW12.But one effector protein specifically expressed in CW12.There were 14 effector proteins expressed in Tq N22 and CW22 and from which 4 effector proteins were found specifically expressed in each of the Tq N22 and CW22.The expression level of DONQB4 was 2.27 times higher in N starvation conditions compared to that occurred in complete nutrient culture.The expression level of DONTI4 under N starvation was 2.63 times higher than that in complete nutrient culture.DOMQL8 was detected only under N starvation conditions.According to the above results,DONQB4,DONTI4 and DOMQL8 with Rx LR were targeted for functional and structural analysis.3.Functional verification and structural analysis of secreted effector proteins in NOD-1 under N starvation conditionThe gene sequences of the above three effectors were retrieved from the NCBI database.The primers for each of the 3 genes were designed using the software Primer and the bacterial expression vector p ET28 a was used for the prokaryotic expression of the above effector genes.The fusion proteins were detected by Western blot.According to the detection results,the proteins were purified by nickel agarose gel affinity chromatography and resulted in two target effectors,DONTI4 and DOMQL8.The effectors obtained,were inoculated singly into N.benthamiana and the pathogenic reactions were observed.By comparing the necrotic spot areas,it was found that DONTI4 induced stronger pathogenicity than DOMQL8.The protein structure analysis showed both the effectors consist mainly of α-helix,β-turn and random coil and the ratios of α-helix were significantly higher than β-turn.The conserved sequence analysis showed totally different pattern at varying sites between the two proteins.The SMART online analysis revealed eight domains in DONTI4 and three domains in DOMQL8.The findings of this study laid a foundation for further functional analysis of these two effectors.There have been a lot of research reports on how N starvation culture can induce the expression of disease-related genes in pathogens and lead to the aggravation of host diseases.However,there are few reports on the mycelium growth and the secretion characteristics of pathogenic proteins produced by the causal fungal organism of P.infestans under N starvation conditions.In this paper,the pathogenicity of the secreted protein generated by the P.infestans NOD-1 of potato was analyzed using N.benthamiana.The mycelia growth characteristics of NOD-1 under N starvation stress were described.And the critical time for NOD-1 to produce strong pathogenic secreted proteins in liquid culture was determined to be 22 d.The Label-free quantitative proteomic analysis techniques combined with bioinformatics analysis showed that under the condition of N starvation stress,several effector genes were induced to express.About 3 up-regulation expression effector genes（including specific expression）with the domain Rx LR structure were screened from the induced genes.Two genes were genetic cloning,prokaryotic expression and their products were purified as DONTI4 and DOMQL8 effector proteins.Functional verification has proven that both the effectors of heterologous expression proteins can result pathogenic reaction in a host.