Cloning And Functional Verification Of Metalaxyl Resistance Candidate Genes In Phytophthora Capsici

Pepper blight caused by Phytophthora capsici can occur in the entire growth period of peppers,posing a huge risk to the cultivation of pepper.Presently,metalaxyl is still a key fungicide to controll the plant oomycete diseases such as the destructive pepper blight around the world.The resistance of the pathogenic oomycete to metalaxyl is becoming serious due to the constant use of this fungicide.To date,the study on the mechanism of metalaxyl resistance development remains unclear.In this study,parental sensitive strain SD1 was used as the WT strain,and a resistant mutant strain SD1-9 was obtained by induction of the fungicide.Used transcriptome sequencing analysis and homologous alignment were obtained five metalaxyl-resistance candidate genes in strain SD1-9,and we cloned them and verified their functionality.Our research would give insight of the molecular mechanism in development of metalaxyl-resistance in P.capsici.Meanwhile,they would also provide references to monitoring and controlling of the metalaxyl resistant isolates of P.capsici,as well as studying the metalaxyl resistant mechanism in other oomycetes in Phytopthora.The main results are as follows:1.The mutant strain SD1-9 having an EC50 value of 326.7 ?g.mL^-1 was obtained by acclimation of the fungicide on a 10% V8 plate containing 10 ?g.mL^-1metalaxyl.The colonies of the SD1-9 on 10% V8 plate without metalaxyl were white,fluffy,and the edges were neat,which was similar to the colony morphology of the parent strain SD1,indicating that the induction of metalaxyl does not change its colony morphology.And under the same conditions,the mutant strain SD1-9 grew slower on the 10% V8 medium without metalaxyl than the WT SD1.The sporangia produced by SD1-9 and SD1 are similar in shape,all of which are oval,oblong,and nearly spherical.The size and shape of the sporangia and the length of the mastoid and stem are different depending on the degree of development.However,the mean values of the strains were not much different,with the length of 38.13³8.98 ?m,a width of26.69²9.74 ?m,aspect ratio of 1.36¹.49 ?m,mastoid height of 4.80⁴.90 ?m,and the stalk length of 37.19³8.47 ?m.In the determination of pathogenicity,the pathogenicity in different plants and leaves showed no significant difference between the parental strain SD1 and the mutant strain SD1-9.However,the virulence of the same strain in different plants and leaves and different fruits is different,and thepathogenicity of eggplant,cucumber and green pepper is the largest.Moreover,the two strains have the same trend in the pathogenicity of the leaves of the bell peppers and peppers leaves under the treatment of metalaxyl at concentrations of 0 ?g.mL^-1,5?g.mL^-1 and 100 ?g.mL^-1.2.Using the sensitive strain SD1 and the resistant mutant strain SD1-9 as experimental materials,we performed transcriptome sequencing analysis.The published P.capsici genome database was used as a reference genome for gene expression difference analysis,functional prediction,GO and KEGG Pathway enrichment analysis.Four genes with significantly up-regulated and down-regulated expression were screened for: XLOC₀20226?down-regulated?,XLOC₀01584?up-regulated?,fgenesh1_pg.C_scaffold₂1000144?up-regulated?,and fgenesh1_pm.C_scaffold₂2000006?up-regulated?.3.The gene fragment RPA190-pc obtained by homologous alignment with the conserved domain of RPA190 gene?RPOLA-N?in P.infestans.The above four candidate genes and RPA190-pc were used as research objects.These five genes were silenced by PEG-mediated protoplast transformation,and RPA190-pc was overexpressed,and seven transformants were obtained.These transformants were used as test materials to study the functions of these five genes.The biological characteristics and pathogenicity of the transformants indicated that these five genes were involved in the growth and pathogenesis of P.capsici,and also involved in the regulation of zoospores released by P.capsici SD1 and SD1-9.Silencing these five genes slowed the growth of P.capsici,and had a significant effect on the mycelial morphology and sporangia production of P.capsici.Stress tests showed that five candidate genes were involved in the adverse effects of P.capsici against external stress conditions.We speculate that this may be related to candidate gene editing small RNA,but further research is needed.4.Metabolites in P.capsici treated with metalaxyl were analyzed by GC-MS metabolomics techniques.The metabolites of mycelium grown in 5 ?g.mL^-1 and 100?g.mL^-1metalaxyl medium were tested.The results showed that under the treatment of metalaxyl,the types and contents of amino acid metabolites of the four strains increased significantly.However,in fatty acid metabolites,the content of stearic acid and hexadecanoic acid in the four strains decreased significantly with the increase of metalaxyl concentration.Fatty acid is the main component of the fungal membrane.The decrease of fatty acid content will affect the fluidity of the cell membrane,leading to impaired cell membrane transport capacity and hindered carbon transport and energy metabolism,resulting in inhibition of P.capsici growth.Data from up-regulation of metabolites indicate increased membrane stiffness associated with pathogen resistance.