Functional Analysis Of P4-ATPase Gene PcATP4 In Phytophora Capsici

Phytophthora capsici is an important plant pathogen belonging to oomycete with a wide host range.In addition to infecting Pepper in the field,it also infects 18 families and 78 other species of plants including Cucurbitaceae,Solanaceae,Cruciferae,Leguminosae,Chenopodiaceae,Caryophyllaceae,and Rosaceae etc.Under suitable growth conditions,P.capsici can cause devastating damage to crops.P-ATPase is a cationic pump that provides energy for cells by hydrolyzing ATP,which is widely distributed in plants,fungi,bacteria and higher eukaryotes.Until now,research on Phytophthora P-ATPase is rarely reported.In this study,we identified and cloned P4-ATPase gene PcATP4 from P.capsici LT1534.The bioinformatics analysis showed that PcATP4 had typical domain and phosphorylation site of the P-ATPase,and the subcellular localization was predicted that was located in the secretory pathway.In the evolutionary relationship,its homologous proteins that exist in fungi,animals and bacteria,but in relationship,the homologous protein of PcATP4 in other oomycetes are far away from fungi and animals.In this study,the expression of PcATP4 gene in the different life and infection stages of P.capsici was detected.It was found that the PcATP4 gene was significantly up-regulated in the sporangium,zoospore,cystospore,cystospore germination and later stages of the infection,and the expression level of cystospore germination stage was 51 times up-regulated.In order to determine the function of PcATP4 in the growth,pathogenicity and resistance,we used the RNAi technology to obtain two silence transformants.Compare to wild type,the growth of silence transformants was decreased,the end of hyphal was deformed,sporangia and oospores could not be formed.We also found that PcATP4 played a role in regulating expression of two sporulation related genes CDC and G-protein.It is interesting to note that the growth rate and morphology of PcATP4 silencing mutant were restored on the substrate with starch as sole carbon source.Moreover,the pathogenicity of silent mutants to tobacco and pepper leaves was significantly decreased.In the infective experiment,the silencing of PcATP4 resulted the decrease of infection ability.In the experiments for the detection of hydrogen peroxide,the accumulation of active oxygen on the leaves was increased after the PcATP4 silencing mutants infected the plants.So,PcATP4 was involved in the pathogenicity of P.capsici.In the experiment of extracellular enzyme determination,it was found that gene PcATP4 negatively regulated peroxidase and laccase secretion showed a positive correlation.In the resistance test,the tolerance of PcATP4-silenced mutants to hydrogen peroxide,CaCl2,NaCl and Sobitol of osmotic stress factors was decreased,the same to cell wall stress factor SDS,while the tolerance to cell wall stress factor CFW was increased,and more sensitive to low temperature stress.The results showed that P4-ATPase gene PcATP4 played an important role in the growth,pathogenicity and responsive to stress of P.capsici.It provides a theoretical basis for further research on the pathogenic mechanism of P.capsici and the prevention and control of P.capsici.