Studies Of The Mechanism Of Interactions Between Phytophthora Capsici And Pyrimorph And Proteomic Profile Of Its Significant Developmental Stages

Phytophthora capsici is an important oomycete pathogen and has a worldwide distribution.It can infect multiple plants,including solanaceous plants,and snap(Phaseolus vulgaris L.)and lima beans.The diseases caused by P.capsici are responsible for important losses each year.Pyrimorph is a novel fungicide used to control P.capsici.The adaptive response of P.capsici to pyrimorph and proteomic profile of its specific developmental stages was investigated.The results were as follows:The proteomic response of P.capsici to pyrimorph was investigated using the iTRAQ technology.The results revealed that 35 proteins were related to the mode of action of pyrimorph against P.capsici.Further bioinformation analysis and biochemical analysis suggested that the mode of action of pyrimorph in P.capsici included the inhibition of cell wall biosynthesis.The baseline sensitivities of 226 P.capsici isolates,tested by mycelial growth inhibition,showed a unimodal distribution with a mean EC50 value of 1.4261±0.4002)mg/ml.Twelve pyrimorph-resistant mutants with stable resistance were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 10-4.The results of biological characteristics and competitive ability revealed that fitness in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates.These results suggest that the risk of P.capsici developing resistance to pyrimorph could be low to moderate.Further study of molecular resistant mechnism of P.capsici to pyrimorph showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable,high resistance in the mutants.Based on this,a real-time polymerase chain reaction(PCR)-based method was developed here.The real-time PCR assay developed will be useful for high-throughput analysis and monitoring the development of pyrimorph resistance in field populations of P.capsici.Here,we present a new phylogenetic analysis of CesA proteins which indicates that oomycete CesA proteins are more closely related to cellulose synthases from plants.In addition,using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity,we have for the first time achieved successful heterologous over-expression and biochemical characterisation of a cellulose synthase(PcCesAl)from P.capsici.Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the sole reaction product.Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to allow for more extensive cellulose synthesis.A global proteomics study was conducted to compare two key asexual life stages of P.capsici,i.e.the mycelium and cysts,to identify stage-specific biochemical processes.A total of 1201 proteins was identified.Seventy-three proteins exhibited different levels of abundance between the mycelium and cysts.The proteins enriched in the mycelium are mainly associated with glycolysis,the tricarboxylic acid(or citric acid)cycle and the pentose phosphate pathway,providing the energy required for thebiosynthesis of cellular building blocks and hyphal growth.In contrast,the proteins that are predominant in cysts are essentially involved in fatty acid degradation,suggesting that the early infection stage of the pathogen relies primarily on fatty acid degradation for energy production.